髓样细胞表达激发受体-1（TREM-1）对重症急性胰腺炎继发感染的诊断价值和分子治疗应用
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摘要
重症急性胰腺炎(severe acute pancreatitis,SAP)是一种病因复杂、发病机制不明、病情凶险、治疗棘手的急腹症,是临床常见的内外科急症。SAP发病率近年呈现不断上升趋势,以加强监护为主的个体化治疗原则在SAP的治疗中已经被广泛接受,临床疗效有明显提高,但病死率仍高达20—30%。
SAP存在着两个死亡高峰,第一个由SIRS引起,多发生在病程第一周,第二个是由感染引起,多发生在1—3周,占病死率的80—90%。如何早期判定感染的发生,成为人们争论的焦点。需要注意到的是,不加选择地应用抗生素,长时间地应用广谱抗生素都会增加菌株耐药和二重感染的概率,延长ICU住院时间,增加死亡率。有胰腺组织坏死不一定有感染,不是手术的绝对指征,但非手术治疗胰腺感染失败并延误手术时机的后果也是致命的。细针穿刺物和临床送检标本的细菌学检查,假性率高且等待时间长(2周左右),以此鉴别胰腺坏死感染与否,临床上很难满意。且多数SAP的发病过程和严重程度往往不是渐进性的,时常会在短时间内急剧变化,造成多器官功能障碍。因此,张圣道、张群华、赵玉沛等教授指出,早期明确诊断SAP感染,尤其是早期识别暴发性胰腺炎是关键。SAP的综合治疗方案已形成共识,但对关键问题:如何早期诊断SAP继发感染,尚存争议。
目前临床上已部分开展通过检测C反应蛋白和降钙素原等,来预测感染和判断预后,但应用价值非常有限。SAP的主要问题之一是尚无单一的血清血指标能准确地早期预示胰腺感染性坏死及精确地预测疾病严重程度。急需找到一个敏感、特异、能即时反映感染过程的指标,监测疾病进展,指导抗生素应用,掌握手术时机。
髓样细胞表达的激发受体-1(Triggering receptor expressed on myeloid cellsTREM-1)属免疫球蛋白超家族。自2001年首次发现,加深了人们对炎症反应的启动和发展过程的认识。TREM-1主要分布在外周血中性粒细胞和单核细胞亚群,这些天然免疫反应的效应细胞上,还选择性地表达在肺泡、肠液及其它体液的吞噬细胞上,能特异性识别病原体的表面受体。TREM-1属跨膜糖蛋白,具有胞外区、跨膜区和较短的胞浆内尾段的保守结构。TREM-1胞外区能特异性与其配体或单克隆抗体结合,跨膜区的赖氨酸残基,可与接头蛋白DAP12跨膜区内的带负电荷的天冬氨酸相耦联,并通过DAP12胞浆区中的免疫受体酪氨酸活化基序(ITAM)来传递活化信号。Bouchon等报道,当革兰阳性菌、阴性菌或真菌所致的急性炎症和肉芽肿性炎症中,以及内毒素(LPS)或其他微生物导致的感染性休克中,感染组织中渗出的中性粒细胞和单核细胞表面的TREM-1表达明显升高。而对于那些非感染引起的免疫复合物炎症反应,如鳞癣、溃疡性结肠炎和脉管炎等,则不会出现TREM-1的表达增加。在体外,用活细菌或其胞壁成分与中性粒细胞和单核细胞共同培养,可诱导免疫细胞表面的TREM-1表达上调。
TREM-1在炎症反应中的表达和诊断价值日益得到重视。TREM-1扩大了细菌及真菌所致感染的炎症反应效应,与下游细胞因子之间形成一个正反馈的自分泌调节回路,影响机体的天然免疫,促使炎症反应增强放大。TREM-1介导的信号转导通路在炎症反应的级联放大和脓毒症的发生中起着关键性的作用。THE NEW ENGLAND医学杂志上报道了通过Western的方法,快速检测患者支气管肺泡灌洗液中的可溶性TREM-1(sTREM-1),有助于确诊或排除细菌性或真菌性肺炎。在脓毒性休克的病人,单核细胞TREM-1表达也不断增高。
TREM-1是炎症反应的潜在分子治疗靶点。根据TREM属于IgSF,TREM-1与IgG具有一定的同源性,有构想TREM-1与IgG融合蛋白综合具有此两种免疫球蛋白的特性,这种融合蛋白能与免疫细胞表面的TREM-1竞争性结合TREM-1配体,从而起抑制活化信号的作用。是目前TREM研究的热点。TREM-1对细菌/真菌引起的感染的预测诊断价值,已逐渐得到公认,其诊断价值和作为分子治疗靶点的意义仍在不断的探索之中。我国对TREM-1的研究却几近空白。
SAP的发病机制,目前仍存在争议。新近提出的炎性介质参与产生的瀑布反应诱发全身炎症反应综合征(SIRS)和多器官功能障碍综合征,丰富了SAP发病机制的学说。有研究检测TREM-1mRNA在轻、重型急性胰腺炎患者及正常人白细胞中表达的差异程度,结果提示TREM-1在急性胰腺炎的发生发展过程中存有重要作用。
本研究拟通过SAP及其继发感染的大鼠和猪模型,分析TREM-1基因的mRNA水平表达情况,揭示TREM-1在SAP和其继发感染中的关键性作用;通过分析SAP患者外周血TREM-1的表达情况,以细菌培养为诊断标准,评估TREM-1作为SAP患者继发腹腔感染指标的诊断价值;构建人TREM1-IgG融合蛋白及大鼠TREM1-IgG融合蛋白的真核表达载体,并在CHO细胞中实现稳定表达;在大肠杆菌中大量表达并纯化大鼠TREM1-IgG融合蛋白;用于治疗SAP大鼠,验证TREM-1作为SAP分子治疗靶点的价值。
更深入地了解TREM-1的信号转导机制,预测诊断SAP感染,把握手术时机,提供分子治疗靶点,为SAP发病机制探讨和综合治疗提供新思路。
一、重症急性胰腺炎大鼠胰腺组织TREM-1基因表达的检测
目的:检测SAP大鼠胰腺组织TREM-1的表达情况。
方法:雄性SD大鼠18只,随机分为假手术组(SO组)、重症急性胰腺炎组(SAP组)和重症急性胰腺炎继发感染组(INP组),各6只。采用胰管逆行注射3%牛磺胆酸钠法构建SAP大鼠模型,造模后6h腹腔注射大肠杆菌悬液构建INP大鼠模型。采用全自动生化分析仪检测血浆淀粉酶,观察各组大鼠胰腺组织病理学变化。并以real-time PCR进行相对定量检测各组大鼠胰腺组织TREM-1的mRNA水平表达情况。
结果:血浆淀粉酶检测和胰腺病理改变,证实造模成功。三组均在造模后24小时,处死大鼠,无菌操作下各取出胰腺组织0.5 g,抽提总mRNA。经逆转录聚合酶链式反应(RT-PCR)扩增出大鼠TREM-1,扩增产物与预期目的基因长度一致,回收纯化目的产物,测序结果与GenBank公布的基因序列完全一致。Real-time PCR进行相对定量检测,INP组胰腺组织TREM-1 mRNA的相对含量显著高于SAP组,而SAP组显著高于SO组(P＜0.05)。
结论:重症急性胰腺炎大鼠胰腺组织TREM-1表达显著增加,提示TREM-1在重症急性胰腺炎的发生发展过程中存有重要作用,且和其严重程度相关。
二、猪重症急性胰腺炎继发感染模型的建立和外周血中TREM-1基因表达的检测
目的:构建猪重症急性胰腺炎继发感染模型,分析SAP猪继发感染前后的外周血中TREM-1的表达情况。
方法:健康家猪6只,3%戊巴比妥钠静脉麻醉后,采用十二指肠镜下胰管内注射牛磺胆酸钠和胰蛋白酶混合液的方法构建SAP模型。造模后24小时内,胰腺CT检查证实造模成功,并留取外周血。即在CT引导下经穿刺针向胰腺坏死部位注入活化大肠杆菌菌液。7天后复查胰腺CT,观察胰腺实质和局部情况,根据胰腺周围积液情况,选择是否再次由CT引导下注射活化的大肠杆菌标准菌株悬液。SAP造模14天后,处死动物,处死前取外周血5ml。观察猪胰腺组织大体及病理学变化。Real-time PCR相对定量检测SAP猪继发感染前后外周血中TREM-1的mRNA水平表达情况。
结果:影象学改变和组织学观察,证实构建猪重症急性胰腺炎继发感染模型成功。抽提外周血中总mRNA,经逆转录聚合酶链式反应(RT-PCR)扩增出猪TREM-1产物与预期目的基因长度一致,回收纯化目的产物,测序结果与GenBank公布的基因序列完全一致。Real-time PCR进行相对定量检测,SAP猪继发感染前后外周血中TREM-1的相对表达量分别为1.51±1.38和4.99±3.28,配对t检验结果表明,外周血中TREM-1mRNA表达水平继发感染后显著高于单纯SAP时(P＜0.05)。
结论:SAP继发感染猪外周血中TREM-1表达显著增加,提示TREM-1参与了SAP发病的全过程。
三、TREM-1作为SAP患者继发腹腔感染指标的诊断价值
目的:克隆了人TREM-1基因的全长,并探讨SAP患者外周血中TREM-1基因表达情况,判断其对SAP患者继发腹腔感染的诊断价值。
方法:收集2006年12月—2007年12月我院消化内科监护室收治的诊断为SAP的患者34例(诊断依据2003年全国胰腺会议确定的急性胰腺炎诊疗指南),入选患者均为发病1-2周后,排除呼吸道、泌尿系统、皮肤表面感染等,高度怀疑继发腹腔感染的患者,收集外周血5ml。抽提外周血中总mRNA,RT-PCR法扩增出人TREM-1,回收纯化目的产物,测序结果正确。检测血清中C反应蛋白(CRP),real-time PCR进行相对定量检测各患者外周血中TREM-1的表达情况。收集患者基本临床资料、治疗方式及预后。以腹水、B超/CT引导下囊肿穿刺物的细菌培养结果或外科手术结果为判断SAP继发腹腔感染的标准,将患者分为感染组和未感染组。通过ROC曲线下面积(AUC)检验TREM-1和CRP在预测诊断指标方面的作用,分别计算其判断SAP继发腹腔感染的特异度和敏感度。
结果:入选的34例SAP患者,其中男性18例,女性16例(男:女=1.125:1);平均年龄48.82±18.08岁。其中明确继发腹腔感染12例,无明确腹腔感染的患者22例。死亡2例,转外科手术3例。SAP继发腹腔感染组的TREM-1相对表达量值显著高于非感染组(P＜0.05)。TREM-1的ROC曲线下面积(AUC)为0.795,判断SAP继发腹腔感染的敏感度和特异度分别为83.8%和81.8%。CRP的ROC曲线下面积(AUC)为0.669,判断SAP继发腹腔感染的敏感度和特异度分别为50%和86.4%。
结论:SAP患者外周血中TREM-1基因表达情况,对SAP继发腹腔感染的诊断能力高于CRP。有望成为临床上SAP患者监测疾病进展,指导抗生素应用,掌握手术时机的指标。
四、TREM1-IgG融合蛋白真核表达载体的构建、表达及鉴定
目的:构建人TREM1-IgG融合蛋白及大鼠TREM1-IgG融合蛋白的真核表达载体,并在CHO细胞中实现稳定表达。
方法:克隆人TREM-1胞外区片段,大鼠TREM-1胞外区片段和人IgG1重链基因Fc恒定区。通过T_4DNA连接酶三片段连接,获得人及大鼠的pcDNA3.1/TREM1-IgG1重组质粒。采用脂质体法,将两个重组质粒分别转染入CHO细胞并G418筛选,进行稳定表达。收集细胞上清液,Protein A-Sepharose CL-4B免疫亲和纯化,快速获取目的蛋白粗品,SDS-PAGE和Western-Blot对蛋白性质进行初步鉴定。
结果:RT-PCR扩增出人TREM-1胞外区、大鼠TREM-1胞外区和人IgG1重链基因Fc恒定区。上述PCR产物与载体pMD-18 T连接后转化大肠杆菌,抽提质粒,酶切测序确认克隆载体构建成功。测序正确的pMD-18 T/人TREM-1胞外区质粒用EcoR和BamHI双酶切,pMD-18 T/大鼠TREM-1胞外区质粒用EcoRI和BamHI双酶切,pMD18T/IgG Fc质粒用BamHI和XhoI双酶切。pcDNA3.1(+)载体质粒分别用EcoR/XhoI和EcoRI/XhoI双酶切。T_4DNAligase行三片段连接,分别构建出人及大鼠的TREM1-IgG1Fc/pcDNA3.1(+)重组质粒,测序结果证实序列、启动子、终止子位置及读码框完全正确。两个重组质粒分别应用lipo2000稳定转染CHO细胞,G418以600μg/ml浓度筛选,RT-PCR表明融合基因在稳定转染的CHO细胞上得到了表达。利用Protein A与目的蛋白IgG1Fc段特异性结合的性质,免疫亲和纯化,快速获取目的蛋白粗品。SDS-PAGE可见66KD左右的牛血清白蛋白和39KD左右的目的蛋白两条带。Western Blot结果证明该蛋白样品可被抗人IgG1 Fc抗体特异性识别,证明该蛋白为IgG融合蛋白。
结论:成功构建了人TREM1-IgG融合蛋白及大鼠TREM1-IgG融合蛋白真核表达载体,并在CHO细胞中稳定表达。
五、大鼠TREM1-IgG融合蛋白原核表达载体的构建、表达及鉴定
目的:构建大鼠TREM1-IgG融合蛋白的原核表达载体,大量表达并纯化鉴定重组大鼠TREM1-IgG融合蛋白。
方法:利用EcoRI和XhoI从重组质粒pcDNA3.1/大鼠TREM1-IgG上切下TREM1-IgG1融合基因,插入原核表达载体质粒pGEX-4T-1相应位点,转化大肠杆菌BL21。IPTG诱导重组融合蛋白表达,超声破碎菌体,Glutathione Sepharose 4B洗脱纯化蛋白,测定融合蛋白浓度,SDS-PAGE、Western-Blot和PMF分析蛋白表达情况。
结果:重组质粒pcDNA3.1/大鼠TREM1-IgG在EcoRI和XhoI双酶切后,电泳证实切下融合基因TREM1-IgG1,插入原核表达载体质粒pGEX-4T-1相应位点,转化BL21后质粒切出分别对应原质粒和目的基因的两条特异性条带,测序证实为阳性克隆。IPTG诱导并纯化后,SDS-PAGE电泳分析表明在66KD左右出现新的蛋白表达条带,大小与带GST的大鼠TREM1-IgG融合蛋白大小相吻合;Western Blot证实该蛋白为IgG融合蛋白;肽质量指纹谱PMF共测得17个匹配的肽段,证实样品为目的蛋白;纯化后融合蛋白浓度为1mg/ml。
结论:成功构建了大鼠TREM1-IgG融合蛋白的原核表达载体,大量表达并纯化出大鼠TREM1-IgG融合蛋白,为进一步研究TREM-1做为SAP分子治疗靶点的价值打下基础。
六、融合蛋白TREM1-IgG治疗SAP大鼠的疗效研究
目的:观察验证融合蛋白TREM1-IgG对SAP大鼠的疗效。
方法:雄性SD大鼠20只,随机分为SAP组、SAP对照蛋白治疗组(GST治疗组)、SAP融合蛋白治疗组(融合蛋白组)、对照组,各5只。采用精氨酸腹腔注射构建SAP大鼠模型,造模成功后3小时,SAP对照蛋白治疗组和SAP融合蛋白治疗组动物分别予以尾静脉注射纯化后的GST蛋白和融合蛋白TREM1-IgG,给药浓度为4mg/kg,SAP组予以500μl生理盐水注射。治疗后24小时处死大鼠,检测血浆淀粉酶、转氨酶及肌酐水平,观察各组大鼠胰腺、肝脏、肺脏组织病理学变化,并对组织损伤评分。
结果:TREM1-IgG融合蛋白治疗24小时后,与SAP组和GST蛋白治疗组相比,肌酐及转氨酶的水平无显著性差异,血清淀粉酶有下降趋势。融合蛋白治疗组的胰腺炎症、出血、坏死和肺脏炎症及脏器总损伤的评分,较其它两组,得到显著改善(P＜0.05)。
结论:融合蛋白TREM1-IgG能有效缓解SAP大鼠的炎症反应,减轻胰腺、肺脏组织损害。在大鼠体内验证了TREM-1做为SAP分子治疗靶点的价值。
通过上述研究,本课题得出以下结论:
1.TREM-1参与了重症急性胰腺炎时的“过度”炎症反应,在SAP继发感染时TREM-1的表达显著升高;
2.SAP患者外周血中TREM-1的基因表达情况的检测,对SAP继发腹腔感染有较好的诊断能力,具有良好的临床应用前景;
3.成功构建了人TREM1-IgG融合蛋白及大鼠TREM1-IgG融合蛋白真核表达载体,并在CHO细胞中稳定表达;
4.成功构建了大鼠TREM1-IgG融合蛋白的原核表达载体,大量表达并纯化出大鼠TREM1-IgG融合蛋白;
5.融合蛋白TREM1-IgG能有效缓解SAP大鼠的炎症反应,减轻胰腺、肺脏组织损害,动物实验证实了TREM-1做为分子治疗靶点对于SAP的应用价值。
小结:该课题揭示了TREM-1在重症急性胰腺炎的发生发展过程中存有重要作用,对SAP继发腹腔感染具有预测诊断价值。构建并表达TREM-1的诱骗受体(大鼠TREM1/人IgG1融合蛋白及人TREM1/人IgG1融合蛋白),通过干预治疗SAP大鼠,验证了TREM-1做为SAP分子治疗靶点的价值。为SAP患者外周血sTREM-1含量检测在临床的应用,和后续寻找鉴定TREM-1的天然配体,奠定了基础。为SAP发病机制的探讨和综合治疗提供新思路。
Severe acute pancreatitis(SAP) is a common emergency in clinic,which is very dangerous and difficult to treat with some unknown characters such as etiology and pathogenesis.The incidence of SAP has the trend of rising in recent years.The principle of individual treatment of SAP has been widely accepted,which markedly enhance the clinical efficacy.However,the mortality rate is still as high as 20%-30%.
There are two deaths peaks in the duration of SAP.The first one caused by SIRS occurs in the course of the first week,and the second is infection,which occurs in 1-3 weeks.Most of the death(80-90%) is caused by infection.The early diagnosis of infection has become the focus of controversy.The most caution in clinic is that non-selective and long time application of broad-spectrum antibiotics will increase the probability of double infection and extend the ICU duration,even increase mortality.Pancreatic tissue necrosis is not necessarily infection,and the surgery is not an absolute indication.The delay caused by the failure of non-surgical treatment in pancreatic infection is fatal.The high false positive rate(FPR) and a long time(about two weeks) of the bacteriological examination of the specimens from the fine-needle aspiration is also dangerous.It is difficult to clinical satisfaction with this method of identification the infected pancreatic necrosis.The majority process SAP is often not gradual and changes in a short time,which always result in multiple organ dysfunction.Hence,Prof.Sheng-dao Zhang,Qun-hua Zhang and Yu-Pei Zhao pointed out that early diagnosis SAP infection is critical in the treatment of SAP. How to identify early diagnosis of secondary infection is the most controversy in the SAP treatment.
C-reactive protein and procalcitonin has been used to predict infection and prognosis in clinic,but the value is very limited.One of the main problems is that there is no single indicator in serum can accurately predict early infection in pancreatic necrosis.A sensitive and specific indicator is required to be found in monitoring disease progression,guiding the use of antibiotics and showing the time of surgery.
The triggering receptor expressed on myeloid cells(TREM) family is a member of the immunoglobulin superfamily and includes at least two activating receptors,namely TREM-1 and TREM-2.The understanding of the process of inflammatory response, included in its start and development,is deeply known since the first discovery of TREM-I in 2001.TREM-1 is mainly distributed in the peripheral blood neutrophil leukocyte and monocytes subsets,which are natural immune response cells.The expression of TREM-1, identifying specifically the surface receptor of pathogens,is also selectively found in the phagocytes in the pulmonary,intestinal fluid and other body fluids.The TREM-1,include extracellular,transmembrane domain and a short cytoplasmic tail section of the conservative structure,is a transmembrane glycoprotein.Engagement of TREMs,after association with the adapter protein DAP12(which contains an immunoreceptor tyrosine-based activation motif),This residue allows pairing with transmembrane adapter proteins,which contain a negatively charged amino acid in the transmembrane domain and a cytoplasmic immunoreceptor tyrosine-based activation motif(ITAM).Bouchon reported that the TREM-1 expression was significantly increased in neutrophilic leukocytes and monocytes surface in infection due to Gram-positive bacteria,fungi or negative bacteria, which caused acute inflammation and granulomatous inflammation,as well as endotoxin (LPS) or other organisms resulting in septic shock.However,in the non-infected immune complex inflammatory response,such as scales psoriasis and ulcerative colitis,it will not appear TREM-1 expression.In vitro,the combined culture with living bacteria or its cell wall composition and neutrophils and monocytes can increased the surface expression of TREM-1.
The diagnostic value of TREM-1 in inflammatory response is receiving increasing attention.TREM-1 expanded the inflammatory response effect caused by bacterial and fungal infections,.There is an autocrine loop of positive feedback formed by downstream cytokines and TREM-1,which will influence the natural immunity and enhance the inflammatory reaction amplification.TREM-1-mediated signal transduction pathway plays a crucial role in the cascade of inflammatory reaction amplification and the incidence of sepsis.Gibot demonstrated that the rapid detection of the soluble TREM-1(sTREM-1) in bronchoalveolar lavage fluid with Western methods,would confirm the judgment of bacterial or fungal pneumonia.In patients with septic shock,the level of TREM-1 expression in monocytes is also high.
TREM- 1 is the potential molecular target in the treatment of inflammation.According the knowledge that the TREM included in IgSF and TREM-1 and IgG had some homology, we demonstrate the possibility of the TREM-1 fusion protein IgG integrated with the two immunoglobulin characteristics.This fusion protein with the surface of immune cells TREM-1 competitive with TREM-1 ligand to inhibit activation of the role of signal.The research in TREM is currently a hot topic.The diagnostic value of TREM-1 on the bacterial / fungal infection has been increasingly recognized,and the exploration as a therapeutic target continues.In China,the research of TREM-1 is almost blank.
There is still in dispute about the pathogenesis of SAP.The newly propose that inflammatory mediators involved in the Waterfall reaction induced systemic inflammatory response syndrome(SIRS) and multiple organ dysfunction,enriched the SAP pathogenesis. A research demonstrated that there is obvious difference of the expression of TREM-1mRNA in interleukin in mild acute pancreatitis(MAP) and severe acute pancreatitis(SAP).The result suggested that the TREM-1 will play an important role in acute pancreatitis.
Firstly,the study is to reveal the importance position of TREM-1 in secondary infection of SAP,through analyzing the mRNA expression level of TREM-1 in SAP rats and pigs.Second,assess the value of TREM-1 as a diagnosis indicator of intra-abdominal secondary infection in SAP patients,examining the TREM-1 expression in peripheral blood,comparing with bacterial culture as the gold standard.Third,respectively in human being and rats,construct TREM1-IgG fusion protein and the protein eukaryotic expression vector,and in CHO cells stability expression is achieved.Fourth,the expression and purify of rat TREM-1/ human IgG1 fusion protein in Escherichia coli are achieved.Last, intervention treatment in SAP rats with decoy receptors was to certify its therapeutic targets value.
1.Detection of TREM-1 expression in pancreatic tissue of SAP rat
Objective:To detect TREM-1 expression in pancreatic tissue of SAP rats and sham-operated rats
Methods:18 male SD rats were randomly divided into sham-operated group,the severe acute pancreatitis(SAP) group and secondary infection group,6 respectively.The construction of the SAP model was successfully achieved through retrograde injection of 3%sodium taurocholate in pancreatic duct of rats.After 6h,the Escherichia coli suspension Construction was injected in abdominal cavity for INP rat model.The histological changes in the pancreas were observed,and the levels of amylase were detected using automatic biochemical analyzer.The expression of TREM-1 mRNA in pancreatic tissue was detected through real-time PCR.
Results:The SAP models were successfully constructed.The rats in three groups were sacrificed in 24 hours.The 0.5 g pancreatic tissue was removed and the total mRNA was extracted.The rats TREM-1 was amplified through the reverse transcription-polymerase chain reaction(RT-PCR).The recovery and purification of the PCR products with the expected length was made,and the same sequence was clarified with the announced GenBank sequence.With the real-time PCR,the mRNA expression of TREM-1 in INP group was significantly higher than that in SAP group(P＜0.05).And the mRNA expression in sham-operated group was lower than that in SAP group.
Conclusion:The TREM-1 expression was significantly increased in SAP and the secondary infection models in rat.These data suggested that TREM-1 might play an important role in the process of severe acute pancreatitis.
2.The construction of secondary infection with severe acute pancreatitis in pig model and the detection of TREM-1 gene expression in peripheral blood
Objective:To construct the model of secondary infection with severe acute pancreatitis in pig and detect the TREM-1 expression in peripheral blood before and after secondary infection.
Methods:3%sodium pentobarbital was injected in abdominal cavity in 6 Pigs.The SAP models were successfully constructed,using injection of the mixture of sodium taurocholate and trypsin in pancreatic duct with duodenal endoscopy.After 24 hours of the procedure,pancreatitis was confirmed with CT scan.Then the blood samples were collected.Immediately the activated E.coil bacterium was injected in necrosis area of the pancreas by the CT-guided needle.7 days later,the local area and the surrounding circumstances were observed with pancreatic CT.According to the scans,weather the injection of the activated strain of E.coli standard suspension was judged.14 days later, the animals were killed.The 5ml blood sample was executed before death.The pancreatic tissue and the pathological changes were observed.The relative quantitative level of TREM-1 mRNA expression in the peripheral blood was detected by real-time PCR before and after secondary infection in pigs.
Results:The constructions of secondary infection model with severe acute pancreatitis in pigs were successful,confirmed by imaging and histological changes.The pig TREM-1 was amplified through the reverse transcription-polymerase chain reaction(RT-PCR).The recovery and purification of the PCR products with the expected length was made,and the same sequence was clarified with the announced GenBank sequence.The relative TREM-1 expression before and after secondary infection with SAP in pig were 1.51±1.38 and 4.99±3.28,respectively.Paired t-test showed that the blood level of TREM-1mRNA after the infection was significantly higher than that before the infection(P＜0.05).
Conclusion:There is a significant increase of TREM-1 in secondary infection with SAP in pig.It is suggested that TREM-1 involved in the pathogenesis of the entire process of SAP.
3.The diagnostic value of TREM-1 as an indicator in patients with intra-abdominal secondary infection
Objective:Cloned human TREM-1 gene,and discuss the presence of soluble TREM-1(sTREM-1) in peripheral blood from patients may be an indicator of secondary infection.
Methods:34 cases of patients after 1-2 weeks admission with SAP(2003 Guideline in China) were selected from 2006 November to 2007 December.All the patients highly suspected abdominal secondary infection,excluding the infection from respiratory tract, urinary tract and skin.The mRNA was extracted in the 5 ml peripheral blood,RT-PCR amplification of TREM-1,the recovery and purification,sequencing results correctly.The level of C-reactive protein(CRP) in serum was tested.The TREM-1 expression was detected with real-time PCR in the peripheral blood.The bacterial culture results of the samples,from B ultrasound / CT guided puncture of the cyst or surgery,was treated as gold standard.Then the patients were divided into infected and non-infected group. Through ROC curve analysis,the diagnosis value of TREM-1 and CRP as indicators to judge the secondary infection were analyzed and the threshold was calculated.
Results:In 34 patients,there was 18 men and 16 women(male:female = 1.125:1), and the average age was 48.82±18.08.Twelve cases were diagnosed as secondary intra-abdominal infection.Two patients died,three cases received surgery.TREM-1 expression in infected and uninfected group had a significant difference(P＜0.05).The area under the ROC curve(AUC) of TREM-1 was 0.795.The sensitivity and specificity were 83.8%and 81.8%.And the AUC of CRP was 0.795,the sensitivity and specificity were 50.0%and 84.6%.
Conclusion:The diagnosis value of TREM-1 expression in the peripheral blood of SAP patients was better than CRP.The combined of these two indictors,can improve the diagnostic efficiency.
4.The constructor of eukaryotic expression vector and expression of TREM1 - IgG fusion protein in human and rat
Objective:To construct the eukaryotic expression vector of TREM1-IgG fusion protein in human and rat and to achieve stable expression in CHO cells.
Methods:Firstly,clone the TREM-1 extracellular region fragment of human,the extracellular domain fragments of rat and the constant region of heavy chain in IgG1-Fc of human,respectively.Second,The gene encoding the mTREM-1/IgGlFc fusion protein were constructed in eukaryotic expression vector pcDNA3.1(+)by means of T-A cloning and subcloning techniques,then was transfected into CHO cells for stable expression. Third,the cell supernatant was collected.Protein A-Sepharose CL-4B-immune affinity purification,and rapid accessed to crude protein.The expression of the fusion protein was detected by SDS-PAGE and Western Blot.
Results:The TREM-1 extracellular region fragment of human,the extracellular domain fragments of rat and the constant region of heavy chain in IgGl-Fc of human gene fragment were synthesized and amplified from the total RNA by RT-PCR.The resulting products were cloned into pMD-18T vector.The pMD-18T/human TREM-1,the pMD-18T/rat TREM-1 and the pMD-18T/human IgGl construct were then transformed into E.coli DH5α,and the gene was sequenced.The eukaryotic expression vector pcDNA3.1(+) colonies were selected and were respectively cut by EcoR / XhoI and EcoRI / XhoI.T4 DNA ligase connects three fragments were constructed.Human and rat TREM1-IgGlFc/pcDNA3.1(+) recombinant plasmid were constructed,sequencing confirmed that sequence,promoter,termination of location and ORF entirely correct.Two recombinant plasmids were stably transfected into CHO cells with lipo2000 and selected with G418.RT-PCR showed that expression of the target gene was found in stably transfected CHO cells lines.The supernatants were harvested,and TREM-IgG fusion protein was purified by protocols including protein A affinity chromatography.In SDS-PAGE assay,about 66 KD bovine serum albumin and 39 KD specific protein were detected.In Western Blot assay,this product could be specifically detected by anti-human IgGl Fc antibody,which confirm it is an IgG fusion protein.
Conclusion:Construction of the Human and rats TREM1-IgGlFc fusion protein eukaryotic expression vector,and stably expressedin CHO cells.
5.Rat TREM1-IgGl fusion protein prokaryotic expression vector construction, expression and identification
Objective:To construct ratTREM1-IgGl fusion protein prokaryotic expression vector, expression and purification of recombinant rat TREM1- IgGl fusion protein.
Methods:Rat TREM1-IgGl fusion gene was cut down from rat TREM1-IgGlFc pcDNA3.1(+) recombinant plasmid by restriction enzymes,EcoRI and XhoI,and inserted into the expression vector corresponding pGEX4T-1 site,and then was transformed into E.coli BL21 where it was induced to express proteins by IPTG(isopropyl-1-thio-b Dgalactopyranoside).The expressed proteins were analyzed through SDS-PAGE,purified through Glutathione Sepharose 4B.Determine the fusion protein concentration.The expression of the fusion protein was detected by SDS-PAGE,Western Blot and PMF.
Results:Rat TREM1-IgGlFc pcDNA3.1(+) recombinant plasmid by restriction enzymes,EcoRI and XhoI.Electrophoresis confirmed that the fusion gene rat TREM1-IgGl was cut down.and inserted into the expression vector corresponding pGEX4T-1 site,and then was transformed into E.coli BL21 where it was induced to express proteins by IPTG.In SDS-PAGE assay,about 66 KD specific protein was detected. In Western Blot assay,this product could be specifically detected by anti-human IgGl Fc antibody,which confirmed it was an IgG fusion protein.17 matching peptide were measured by peptide mass fingerprint PMF,which confirmed that samples were the purpose proteins.And the concentration of the purified fusion protein was 1 mg / ml.
Conclusion:Construction rat TREM1- IgC.l fusion protein prokaryotic expression vector,expression and purification of recombinant rat TREM1- IgG.l fusion protein. Laying the foundation for further study of its as a therapeutic target molecule SAP.
6.The efficacy study of the fusion TREM1-IgG protein in the treatment of SAP rats
Objective:To observe the effect of the fusion TREM1-IgG protein in the treatment of SAP rats.
Methods:20 male SD rats were randomly divided into SAP group(group A),the control treatment(GST) group(group B) an,the fusion protein therapy group(group C) and the control group(n=5).The SAP models in rats were successfully constructed by intraperitoneal arginine injection.After six hours,the treatment was given.The GST protein and fusion protein were respectively injected in tail vein in Group B and C.The concentration was 4mg/kg.In group A,500μl saline was injected.After 24 hours,all the rats were killed.And the levels of amylase,aminopherase and creatinine in serum were detected.The histological changes were observed and scored in pancreas,liver and lung.
Results:24 hours after the treatment,there was no significant difference in the level of aminotransferase and creatinine among GST group,TREM1-IgG fusion protein group and SAP group,The level of serum amylase in TREM1-IgG fusion protein group had a downward trend.In TREM1-IgG fusion protein group,the pathology scores in pancreatic inflammation,bleeding,necrosis,lung inflammation and total organ damage had significantly improved than the other two groups(P＜0.05).
Conclusion:The fusion TREM1-IgG protein can effectively alleviate the inflammatory response and reduce damage to pancreas and lung.In SAP rats,it was proved that TREM-1 as a therapeutic target molecule in SAP is possible.
Through this study,the subjects come to the following conclusions:
1.TREM-1 involved in severe acute pancreatitis at the "excessive" inflammatory response,the expression level of TREM-1 was obviously high when the secondary infection happened.
2.The TREM-1 gene expression in the peripheral blood was a non-invasive,rapid and economic indictor in intra-abdominal secondary infection of SAP patients.The use of TREM-1 was prospected to judge the secondary infection and the time of surgery intervene.
3.Successfully constructed the eukaryotic expression vector of human TREM1-IgG fusion protein and achieved stable expression in CHO cells.
4.Successfully constructed the prokaryotic expression vector of rat TREM1-IgG fusion protein and achieved stable expression and purification.
5.The fusion TREM1-IgG protein can effectively alleviate the inflammatory response and reduce damage to pancreas and lung.In SAP rats,it was proved that TREM-1 as a therapeutic target molecule in SAP is possible.
Summary:The subject demonstrated that TREM-1 played an important role in the process of severe acute pancreatitis and had the diagnosis value in the intra-abdominal secondary infection.Construction and expression of TREM-1 / IgGl fusion protein eukaryotic and prokaryotic expression vector,through the intervention of SAP rats proved TREM-1 as a therapeutic target molecule SAP value.To laying the foundation for further study of follow-up search the natural ligand of TREM-1,and providing new ideas of SAP pathogenesis and comprehensive treatment.
SAP存在着两个死亡高峰,第一个由SIRS引起,多发生在病程第一周,第二个是由感染引起,多发生在1—3周,占病死率的80—90%。如何早期判定感染的发生,成为人们争论的焦点。需要注意到的是,不加选择地应用抗生素,长时间地应用广谱抗生素都会增加菌株耐药和二重感染的概率,延长ICU住院时间,增加死亡率。有胰腺组织坏死不一定有感染,不是手术的绝对指征,但非手术治疗胰腺感染失败并延误手术时机的后果也是致命的。细针穿刺物和临床送检标本的细菌学检查,假性率高且等待时间长(2周左右),以此鉴别胰腺坏死感染与否,临床上很难满意。且多数SAP的发病过程和严重程度往往不是渐进性的,时常会在短时间内急剧变化,造成多器官功能障碍。因此,张圣道、张群华、赵玉沛等教授指出,早期明确诊断SAP感染,尤其是早期识别暴发性胰腺炎是关键。SAP的综合治疗方案已形成共识,但对关键问题:如何早期诊断SAP继发感染,尚存争议。
目前临床上已部分开展通过检测C反应蛋白和降钙素原等,来预测感染和判断预后,但应用价值非常有限。SAP的主要问题之一是尚无单一的血清血指标能准确地早期预示胰腺感染性坏死及精确地预测疾病严重程度。急需找到一个敏感、特异、能即时反映感染过程的指标,监测疾病进展,指导抗生素应用,掌握手术时机。
髓样细胞表达的激发受体-1(Triggering receptor expressed on myeloid cellsTREM-1)属免疫球蛋白超家族。自2001年首次发现,加深了人们对炎症反应的启动和发展过程的认识。TREM-1主要分布在外周血中性粒细胞和单核细胞亚群,这些天然免疫反应的效应细胞上,还选择性地表达在肺泡、肠液及其它体液的吞噬细胞上,能特异性识别病原体的表面受体。TREM-1属跨膜糖蛋白,具有胞外区、跨膜区和较短的胞浆内尾段的保守结构。TREM-1胞外区能特异性与其配体或单克隆抗体结合,跨膜区的赖氨酸残基,可与接头蛋白DAP12跨膜区内的带负电荷的天冬氨酸相耦联,并通过DAP12胞浆区中的免疫受体酪氨酸活化基序(ITAM)来传递活化信号。Bouchon等报道,当革兰阳性菌、阴性菌或真菌所致的急性炎症和肉芽肿性炎症中,以及内毒素(LPS)或其他微生物导致的感染性休克中,感染组织中渗出的中性粒细胞和单核细胞表面的TREM-1表达明显升高。而对于那些非感染引起的免疫复合物炎症反应,如鳞癣、溃疡性结肠炎和脉管炎等,则不会出现TREM-1的表达增加。在体外,用活细菌或其胞壁成分与中性粒细胞和单核细胞共同培养,可诱导免疫细胞表面的TREM-1表达上调。
TREM-1在炎症反应中的表达和诊断价值日益得到重视。TREM-1扩大了细菌及真菌所致感染的炎症反应效应,与下游细胞因子之间形成一个正反馈的自分泌调节回路,影响机体的天然免疫,促使炎症反应增强放大。TREM-1介导的信号转导通路在炎症反应的级联放大和脓毒症的发生中起着关键性的作用。THE NEW ENGLAND医学杂志上报道了通过Western的方法,快速检测患者支气管肺泡灌洗液中的可溶性TREM-1(sTREM-1),有助于确诊或排除细菌性或真菌性肺炎。在脓毒性休克的病人,单核细胞TREM-1表达也不断增高。
TREM-1是炎症反应的潜在分子治疗靶点。根据TREM属于IgSF,TREM-1与IgG具有一定的同源性,有构想TREM-1与IgG融合蛋白综合具有此两种免疫球蛋白的特性,这种融合蛋白能与免疫细胞表面的TREM-1竞争性结合TREM-1配体,从而起抑制活化信号的作用。是目前TREM研究的热点。TREM-1对细菌/真菌引起的感染的预测诊断价值,已逐渐得到公认,其诊断价值和作为分子治疗靶点的意义仍在不断的探索之中。我国对TREM-1的研究却几近空白。
SAP的发病机制,目前仍存在争议。新近提出的炎性介质参与产生的瀑布反应诱发全身炎症反应综合征(SIRS)和多器官功能障碍综合征,丰富了SAP发病机制的学说。有研究检测TREM-1mRNA在轻、重型急性胰腺炎患者及正常人白细胞中表达的差异程度,结果提示TREM-1在急性胰腺炎的发生发展过程中存有重要作用。
本研究拟通过SAP及其继发感染的大鼠和猪模型,分析TREM-1基因的mRNA水平表达情况,揭示TREM-1在SAP和其继发感染中的关键性作用;通过分析SAP患者外周血TREM-1的表达情况,以细菌培养为诊断标准,评估TREM-1作为SAP患者继发腹腔感染指标的诊断价值;构建人TREM1-IgG融合蛋白及大鼠TREM1-IgG融合蛋白的真核表达载体,并在CHO细胞中实现稳定表达;在大肠杆菌中大量表达并纯化大鼠TREM1-IgG融合蛋白;用于治疗SAP大鼠,验证TREM-1作为SAP分子治疗靶点的价值。
更深入地了解TREM-1的信号转导机制,预测诊断SAP感染,把握手术时机,提供分子治疗靶点,为SAP发病机制探讨和综合治疗提供新思路。
一、重症急性胰腺炎大鼠胰腺组织TREM-1基因表达的检测
目的:检测SAP大鼠胰腺组织TREM-1的表达情况。
方法:雄性SD大鼠18只,随机分为假手术组(SO组)、重症急性胰腺炎组(SAP组)和重症急性胰腺炎继发感染组(INP组),各6只。采用胰管逆行注射3%牛磺胆酸钠法构建SAP大鼠模型,造模后6h腹腔注射大肠杆菌悬液构建INP大鼠模型。采用全自动生化分析仪检测血浆淀粉酶,观察各组大鼠胰腺组织病理学变化。并以real-time PCR进行相对定量检测各组大鼠胰腺组织TREM-1的mRNA水平表达情况。
结果:血浆淀粉酶检测和胰腺病理改变,证实造模成功。三组均在造模后24小时,处死大鼠,无菌操作下各取出胰腺组织0.5 g,抽提总mRNA。经逆转录聚合酶链式反应(RT-PCR)扩增出大鼠TREM-1,扩增产物与预期目的基因长度一致,回收纯化目的产物,测序结果与GenBank公布的基因序列完全一致。Real-time PCR进行相对定量检测,INP组胰腺组织TREM-1 mRNA的相对含量显著高于SAP组,而SAP组显著高于SO组(P＜0.05)。
结论:重症急性胰腺炎大鼠胰腺组织TREM-1表达显著增加,提示TREM-1在重症急性胰腺炎的发生发展过程中存有重要作用,且和其严重程度相关。
二、猪重症急性胰腺炎继发感染模型的建立和外周血中TREM-1基因表达的检测
目的:构建猪重症急性胰腺炎继发感染模型,分析SAP猪继发感染前后的外周血中TREM-1的表达情况。
方法:健康家猪6只,3%戊巴比妥钠静脉麻醉后,采用十二指肠镜下胰管内注射牛磺胆酸钠和胰蛋白酶混合液的方法构建SAP模型。造模后24小时内,胰腺CT检查证实造模成功,并留取外周血。即在CT引导下经穿刺针向胰腺坏死部位注入活化大肠杆菌菌液。7天后复查胰腺CT,观察胰腺实质和局部情况,根据胰腺周围积液情况,选择是否再次由CT引导下注射活化的大肠杆菌标准菌株悬液。SAP造模14天后,处死动物,处死前取外周血5ml。观察猪胰腺组织大体及病理学变化。Real-time PCR相对定量检测SAP猪继发感染前后外周血中TREM-1的mRNA水平表达情况。
结果:影象学改变和组织学观察,证实构建猪重症急性胰腺炎继发感染模型成功。抽提外周血中总mRNA,经逆转录聚合酶链式反应(RT-PCR)扩增出猪TREM-1产物与预期目的基因长度一致,回收纯化目的产物,测序结果与GenBank公布的基因序列完全一致。Real-time PCR进行相对定量检测,SAP猪继发感染前后外周血中TREM-1的相对表达量分别为1.51±1.38和4.99±3.28,配对t检验结果表明,外周血中TREM-1mRNA表达水平继发感染后显著高于单纯SAP时(P＜0.05)。
结论:SAP继发感染猪外周血中TREM-1表达显著增加,提示TREM-1参与了SAP发病的全过程。
三、TREM-1作为SAP患者继发腹腔感染指标的诊断价值
目的:克隆了人TREM-1基因的全长,并探讨SAP患者外周血中TREM-1基因表达情况,判断其对SAP患者继发腹腔感染的诊断价值。
方法:收集2006年12月—2007年12月我院消化内科监护室收治的诊断为SAP的患者34例(诊断依据2003年全国胰腺会议确定的急性胰腺炎诊疗指南),入选患者均为发病1-2周后,排除呼吸道、泌尿系统、皮肤表面感染等,高度怀疑继发腹腔感染的患者,收集外周血5ml。抽提外周血中总mRNA,RT-PCR法扩增出人TREM-1,回收纯化目的产物,测序结果正确。检测血清中C反应蛋白(CRP),real-time PCR进行相对定量检测各患者外周血中TREM-1的表达情况。收集患者基本临床资料、治疗方式及预后。以腹水、B超/CT引导下囊肿穿刺物的细菌培养结果或外科手术结果为判断SAP继发腹腔感染的标准,将患者分为感染组和未感染组。通过ROC曲线下面积(AUC)检验TREM-1和CRP在预测诊断指标方面的作用,分别计算其判断SAP继发腹腔感染的特异度和敏感度。
结果:入选的34例SAP患者,其中男性18例,女性16例(男:女=1.125:1);平均年龄48.82±18.08岁。其中明确继发腹腔感染12例,无明确腹腔感染的患者22例。死亡2例,转外科手术3例。SAP继发腹腔感染组的TREM-1相对表达量值显著高于非感染组(P＜0.05)。TREM-1的ROC曲线下面积(AUC)为0.795,判断SAP继发腹腔感染的敏感度和特异度分别为83.8%和81.8%。CRP的ROC曲线下面积(AUC)为0.669,判断SAP继发腹腔感染的敏感度和特异度分别为50%和86.4%。
结论:SAP患者外周血中TREM-1基因表达情况,对SAP继发腹腔感染的诊断能力高于CRP。有望成为临床上SAP患者监测疾病进展,指导抗生素应用,掌握手术时机的指标。
四、TREM1-IgG融合蛋白真核表达载体的构建、表达及鉴定
目的:构建人TREM1-IgG融合蛋白及大鼠TREM1-IgG融合蛋白的真核表达载体,并在CHO细胞中实现稳定表达。
方法:克隆人TREM-1胞外区片段,大鼠TREM-1胞外区片段和人IgG1重链基因Fc恒定区。通过T_4DNA连接酶三片段连接,获得人及大鼠的pcDNA3.1/TREM1-IgG1重组质粒。采用脂质体法,将两个重组质粒分别转染入CHO细胞并G418筛选,进行稳定表达。收集细胞上清液,Protein A-Sepharose CL-4B免疫亲和纯化,快速获取目的蛋白粗品,SDS-PAGE和Western-Blot对蛋白性质进行初步鉴定。
结果:RT-PCR扩增出人TREM-1胞外区、大鼠TREM-1胞外区和人IgG1重链基因Fc恒定区。上述PCR产物与载体pMD-18 T连接后转化大肠杆菌,抽提质粒,酶切测序确认克隆载体构建成功。测序正确的pMD-18 T/人TREM-1胞外区质粒用EcoR和BamHI双酶切,pMD-18 T/大鼠TREM-1胞外区质粒用EcoRI和BamHI双酶切,pMD18T/IgG Fc质粒用BamHI和XhoI双酶切。pcDNA3.1(+)载体质粒分别用EcoR/XhoI和EcoRI/XhoI双酶切。T_4DNAligase行三片段连接,分别构建出人及大鼠的TREM1-IgG1Fc/pcDNA3.1(+)重组质粒,测序结果证实序列、启动子、终止子位置及读码框完全正确。两个重组质粒分别应用lipo2000稳定转染CHO细胞,G418以600μg/ml浓度筛选,RT-PCR表明融合基因在稳定转染的CHO细胞上得到了表达。利用Protein A与目的蛋白IgG1Fc段特异性结合的性质,免疫亲和纯化,快速获取目的蛋白粗品。SDS-PAGE可见66KD左右的牛血清白蛋白和39KD左右的目的蛋白两条带。Western Blot结果证明该蛋白样品可被抗人IgG1 Fc抗体特异性识别,证明该蛋白为IgG融合蛋白。
结论:成功构建了人TREM1-IgG融合蛋白及大鼠TREM1-IgG融合蛋白真核表达载体,并在CHO细胞中稳定表达。
五、大鼠TREM1-IgG融合蛋白原核表达载体的构建、表达及鉴定
目的:构建大鼠TREM1-IgG融合蛋白的原核表达载体,大量表达并纯化鉴定重组大鼠TREM1-IgG融合蛋白。
方法:利用EcoRI和XhoI从重组质粒pcDNA3.1/大鼠TREM1-IgG上切下TREM1-IgG1融合基因,插入原核表达载体质粒pGEX-4T-1相应位点,转化大肠杆菌BL21。IPTG诱导重组融合蛋白表达,超声破碎菌体,Glutathione Sepharose 4B洗脱纯化蛋白,测定融合蛋白浓度,SDS-PAGE、Western-Blot和PMF分析蛋白表达情况。
结果:重组质粒pcDNA3.1/大鼠TREM1-IgG在EcoRI和XhoI双酶切后,电泳证实切下融合基因TREM1-IgG1,插入原核表达载体质粒pGEX-4T-1相应位点,转化BL21后质粒切出分别对应原质粒和目的基因的两条特异性条带,测序证实为阳性克隆。IPTG诱导并纯化后,SDS-PAGE电泳分析表明在66KD左右出现新的蛋白表达条带,大小与带GST的大鼠TREM1-IgG融合蛋白大小相吻合;Western Blot证实该蛋白为IgG融合蛋白;肽质量指纹谱PMF共测得17个匹配的肽段,证实样品为目的蛋白;纯化后融合蛋白浓度为1mg/ml。
结论:成功构建了大鼠TREM1-IgG融合蛋白的原核表达载体,大量表达并纯化出大鼠TREM1-IgG融合蛋白,为进一步研究TREM-1做为SAP分子治疗靶点的价值打下基础。
六、融合蛋白TREM1-IgG治疗SAP大鼠的疗效研究
目的:观察验证融合蛋白TREM1-IgG对SAP大鼠的疗效。
方法:雄性SD大鼠20只,随机分为SAP组、SAP对照蛋白治疗组(GST治疗组)、SAP融合蛋白治疗组(融合蛋白组)、对照组,各5只。采用精氨酸腹腔注射构建SAP大鼠模型,造模成功后3小时,SAP对照蛋白治疗组和SAP融合蛋白治疗组动物分别予以尾静脉注射纯化后的GST蛋白和融合蛋白TREM1-IgG,给药浓度为4mg/kg,SAP组予以500μl生理盐水注射。治疗后24小时处死大鼠,检测血浆淀粉酶、转氨酶及肌酐水平,观察各组大鼠胰腺、肝脏、肺脏组织病理学变化,并对组织损伤评分。
结果:TREM1-IgG融合蛋白治疗24小时后,与SAP组和GST蛋白治疗组相比,肌酐及转氨酶的水平无显著性差异,血清淀粉酶有下降趋势。融合蛋白治疗组的胰腺炎症、出血、坏死和肺脏炎症及脏器总损伤的评分,较其它两组,得到显著改善(P＜0.05)。
结论:融合蛋白TREM1-IgG能有效缓解SAP大鼠的炎症反应,减轻胰腺、肺脏组织损害。在大鼠体内验证了TREM-1做为SAP分子治疗靶点的价值。
通过上述研究,本课题得出以下结论:
1.TREM-1参与了重症急性胰腺炎时的“过度”炎症反应,在SAP继发感染时TREM-1的表达显著升高;
2.SAP患者外周血中TREM-1的基因表达情况的检测,对SAP继发腹腔感染有较好的诊断能力,具有良好的临床应用前景;
3.成功构建了人TREM1-IgG融合蛋白及大鼠TREM1-IgG融合蛋白真核表达载体,并在CHO细胞中稳定表达;
4.成功构建了大鼠TREM1-IgG融合蛋白的原核表达载体,大量表达并纯化出大鼠TREM1-IgG融合蛋白;
5.融合蛋白TREM1-IgG能有效缓解SAP大鼠的炎症反应,减轻胰腺、肺脏组织损害,动物实验证实了TREM-1做为分子治疗靶点对于SAP的应用价值。
小结:该课题揭示了TREM-1在重症急性胰腺炎的发生发展过程中存有重要作用,对SAP继发腹腔感染具有预测诊断价值。构建并表达TREM-1的诱骗受体(大鼠TREM1/人IgG1融合蛋白及人TREM1/人IgG1融合蛋白),通过干预治疗SAP大鼠,验证了TREM-1做为SAP分子治疗靶点的价值。为SAP患者外周血sTREM-1含量检测在临床的应用,和后续寻找鉴定TREM-1的天然配体,奠定了基础。为SAP发病机制的探讨和综合治疗提供新思路。
Severe acute pancreatitis(SAP) is a common emergency in clinic,which is very dangerous and difficult to treat with some unknown characters such as etiology and pathogenesis.The incidence of SAP has the trend of rising in recent years.The principle of individual treatment of SAP has been widely accepted,which markedly enhance the clinical efficacy.However,the mortality rate is still as high as 20%-30%.
There are two deaths peaks in the duration of SAP.The first one caused by SIRS occurs in the course of the first week,and the second is infection,which occurs in 1-3 weeks.Most of the death(80-90%) is caused by infection.The early diagnosis of infection has become the focus of controversy.The most caution in clinic is that non-selective and long time application of broad-spectrum antibiotics will increase the probability of double infection and extend the ICU duration,even increase mortality.Pancreatic tissue necrosis is not necessarily infection,and the surgery is not an absolute indication.The delay caused by the failure of non-surgical treatment in pancreatic infection is fatal.The high false positive rate(FPR) and a long time(about two weeks) of the bacteriological examination of the specimens from the fine-needle aspiration is also dangerous.It is difficult to clinical satisfaction with this method of identification the infected pancreatic necrosis.The majority process SAP is often not gradual and changes in a short time,which always result in multiple organ dysfunction.Hence,Prof.Sheng-dao Zhang,Qun-hua Zhang and Yu-Pei Zhao pointed out that early diagnosis SAP infection is critical in the treatment of SAP. How to identify early diagnosis of secondary infection is the most controversy in the SAP treatment.
C-reactive protein and procalcitonin has been used to predict infection and prognosis in clinic,but the value is very limited.One of the main problems is that there is no single indicator in serum can accurately predict early infection in pancreatic necrosis.A sensitive and specific indicator is required to be found in monitoring disease progression,guiding the use of antibiotics and showing the time of surgery.
The triggering receptor expressed on myeloid cells(TREM) family is a member of the immunoglobulin superfamily and includes at least two activating receptors,namely TREM-1 and TREM-2.The understanding of the process of inflammatory response, included in its start and development,is deeply known since the first discovery of TREM-I in 2001.TREM-1 is mainly distributed in the peripheral blood neutrophil leukocyte and monocytes subsets,which are natural immune response cells.The expression of TREM-1, identifying specifically the surface receptor of pathogens,is also selectively found in the phagocytes in the pulmonary,intestinal fluid and other body fluids.The TREM-1,include extracellular,transmembrane domain and a short cytoplasmic tail section of the conservative structure,is a transmembrane glycoprotein.Engagement of TREMs,after association with the adapter protein DAP12(which contains an immunoreceptor tyrosine-based activation motif),This residue allows pairing with transmembrane adapter proteins,which contain a negatively charged amino acid in the transmembrane domain and a cytoplasmic immunoreceptor tyrosine-based activation motif(ITAM).Bouchon reported that the TREM-1 expression was significantly increased in neutrophilic leukocytes and monocytes surface in infection due to Gram-positive bacteria,fungi or negative bacteria, which caused acute inflammation and granulomatous inflammation,as well as endotoxin (LPS) or other organisms resulting in septic shock.However,in the non-infected immune complex inflammatory response,such as scales psoriasis and ulcerative colitis,it will not appear TREM-1 expression.In vitro,the combined culture with living bacteria or its cell wall composition and neutrophils and monocytes can increased the surface expression of TREM-1.
The diagnostic value of TREM-1 in inflammatory response is receiving increasing attention.TREM-1 expanded the inflammatory response effect caused by bacterial and fungal infections,.There is an autocrine loop of positive feedback formed by downstream cytokines and TREM-1,which will influence the natural immunity and enhance the inflammatory reaction amplification.TREM-1-mediated signal transduction pathway plays a crucial role in the cascade of inflammatory reaction amplification and the incidence of sepsis.Gibot demonstrated that the rapid detection of the soluble TREM-1(sTREM-1) in bronchoalveolar lavage fluid with Western methods,would confirm the judgment of bacterial or fungal pneumonia.In patients with septic shock,the level of TREM-1 expression in monocytes is also high.
TREM- 1 is the potential molecular target in the treatment of inflammation.According the knowledge that the TREM included in IgSF and TREM-1 and IgG had some homology, we demonstrate the possibility of the TREM-1 fusion protein IgG integrated with the two immunoglobulin characteristics.This fusion protein with the surface of immune cells TREM-1 competitive with TREM-1 ligand to inhibit activation of the role of signal.The research in TREM is currently a hot topic.The diagnostic value of TREM-1 on the bacterial / fungal infection has been increasingly recognized,and the exploration as a therapeutic target continues.In China,the research of TREM-1 is almost blank.
There is still in dispute about the pathogenesis of SAP.The newly propose that inflammatory mediators involved in the Waterfall reaction induced systemic inflammatory response syndrome(SIRS) and multiple organ dysfunction,enriched the SAP pathogenesis. A research demonstrated that there is obvious difference of the expression of TREM-1mRNA in interleukin in mild acute pancreatitis(MAP) and severe acute pancreatitis(SAP).The result suggested that the TREM-1 will play an important role in acute pancreatitis.
Firstly,the study is to reveal the importance position of TREM-1 in secondary infection of SAP,through analyzing the mRNA expression level of TREM-1 in SAP rats and pigs.Second,assess the value of TREM-1 as a diagnosis indicator of intra-abdominal secondary infection in SAP patients,examining the TREM-1 expression in peripheral blood,comparing with bacterial culture as the gold standard.Third,respectively in human being and rats,construct TREM1-IgG fusion protein and the protein eukaryotic expression vector,and in CHO cells stability expression is achieved.Fourth,the expression and purify of rat TREM-1/ human IgG1 fusion protein in Escherichia coli are achieved.Last, intervention treatment in SAP rats with decoy receptors was to certify its therapeutic targets value.
1.Detection of TREM-1 expression in pancreatic tissue of SAP rat
Objective:To detect TREM-1 expression in pancreatic tissue of SAP rats and sham-operated rats
Methods:18 male SD rats were randomly divided into sham-operated group,the severe acute pancreatitis(SAP) group and secondary infection group,6 respectively.The construction of the SAP model was successfully achieved through retrograde injection of 3%sodium taurocholate in pancreatic duct of rats.After 6h,the Escherichia coli suspension Construction was injected in abdominal cavity for INP rat model.The histological changes in the pancreas were observed,and the levels of amylase were detected using automatic biochemical analyzer.The expression of TREM-1 mRNA in pancreatic tissue was detected through real-time PCR.
Results:The SAP models were successfully constructed.The rats in three groups were sacrificed in 24 hours.The 0.5 g pancreatic tissue was removed and the total mRNA was extracted.The rats TREM-1 was amplified through the reverse transcription-polymerase chain reaction(RT-PCR).The recovery and purification of the PCR products with the expected length was made,and the same sequence was clarified with the announced GenBank sequence.With the real-time PCR,the mRNA expression of TREM-1 in INP group was significantly higher than that in SAP group(P＜0.05).And the mRNA expression in sham-operated group was lower than that in SAP group.
Conclusion:The TREM-1 expression was significantly increased in SAP and the secondary infection models in rat.These data suggested that TREM-1 might play an important role in the process of severe acute pancreatitis.
2.The construction of secondary infection with severe acute pancreatitis in pig model and the detection of TREM-1 gene expression in peripheral blood
Objective:To construct the model of secondary infection with severe acute pancreatitis in pig and detect the TREM-1 expression in peripheral blood before and after secondary infection.
Methods:3%sodium pentobarbital was injected in abdominal cavity in 6 Pigs.The SAP models were successfully constructed,using injection of the mixture of sodium taurocholate and trypsin in pancreatic duct with duodenal endoscopy.After 24 hours of the procedure,pancreatitis was confirmed with CT scan.Then the blood samples were collected.Immediately the activated E.coil bacterium was injected in necrosis area of the pancreas by the CT-guided needle.7 days later,the local area and the surrounding circumstances were observed with pancreatic CT.According to the scans,weather the injection of the activated strain of E.coli standard suspension was judged.14 days later, the animals were killed.The 5ml blood sample was executed before death.The pancreatic tissue and the pathological changes were observed.The relative quantitative level of TREM-1 mRNA expression in the peripheral blood was detected by real-time PCR before and after secondary infection in pigs.
Results:The constructions of secondary infection model with severe acute pancreatitis in pigs were successful,confirmed by imaging and histological changes.The pig TREM-1 was amplified through the reverse transcription-polymerase chain reaction(RT-PCR).The recovery and purification of the PCR products with the expected length was made,and the same sequence was clarified with the announced GenBank sequence.The relative TREM-1 expression before and after secondary infection with SAP in pig were 1.51±1.38 and 4.99±3.28,respectively.Paired t-test showed that the blood level of TREM-1mRNA after the infection was significantly higher than that before the infection(P＜0.05).
Conclusion:There is a significant increase of TREM-1 in secondary infection with SAP in pig.It is suggested that TREM-1 involved in the pathogenesis of the entire process of SAP.
3.The diagnostic value of TREM-1 as an indicator in patients with intra-abdominal secondary infection
Objective:Cloned human TREM-1 gene,and discuss the presence of soluble TREM-1(sTREM-1) in peripheral blood from patients may be an indicator of secondary infection.
Methods:34 cases of patients after 1-2 weeks admission with SAP(2003 Guideline in China) were selected from 2006 November to 2007 December.All the patients highly suspected abdominal secondary infection,excluding the infection from respiratory tract, urinary tract and skin.The mRNA was extracted in the 5 ml peripheral blood,RT-PCR amplification of TREM-1,the recovery and purification,sequencing results correctly.The level of C-reactive protein(CRP) in serum was tested.The TREM-1 expression was detected with real-time PCR in the peripheral blood.The bacterial culture results of the samples,from B ultrasound / CT guided puncture of the cyst or surgery,was treated as gold standard.Then the patients were divided into infected and non-infected group. Through ROC curve analysis,the diagnosis value of TREM-1 and CRP as indicators to judge the secondary infection were analyzed and the threshold was calculated.
Results:In 34 patients,there was 18 men and 16 women(male:female = 1.125:1), and the average age was 48.82±18.08.Twelve cases were diagnosed as secondary intra-abdominal infection.Two patients died,three cases received surgery.TREM-1 expression in infected and uninfected group had a significant difference(P＜0.05).The area under the ROC curve(AUC) of TREM-1 was 0.795.The sensitivity and specificity were 83.8%and 81.8%.And the AUC of CRP was 0.795,the sensitivity and specificity were 50.0%and 84.6%.
Conclusion:The diagnosis value of TREM-1 expression in the peripheral blood of SAP patients was better than CRP.The combined of these two indictors,can improve the diagnostic efficiency.
4.The constructor of eukaryotic expression vector and expression of TREM1 - IgG fusion protein in human and rat
Objective:To construct the eukaryotic expression vector of TREM1-IgG fusion protein in human and rat and to achieve stable expression in CHO cells.
Methods:Firstly,clone the TREM-1 extracellular region fragment of human,the extracellular domain fragments of rat and the constant region of heavy chain in IgG1-Fc of human,respectively.Second,The gene encoding the mTREM-1/IgGlFc fusion protein were constructed in eukaryotic expression vector pcDNA3.1(+)by means of T-A cloning and subcloning techniques,then was transfected into CHO cells for stable expression. Third,the cell supernatant was collected.Protein A-Sepharose CL-4B-immune affinity purification,and rapid accessed to crude protein.The expression of the fusion protein was detected by SDS-PAGE and Western Blot.
Results:The TREM-1 extracellular region fragment of human,the extracellular domain fragments of rat and the constant region of heavy chain in IgGl-Fc of human gene fragment were synthesized and amplified from the total RNA by RT-PCR.The resulting products were cloned into pMD-18T vector.The pMD-18T/human TREM-1,the pMD-18T/rat TREM-1 and the pMD-18T/human IgGl construct were then transformed into E.coli DH5α,and the gene was sequenced.The eukaryotic expression vector pcDNA3.1(+) colonies were selected and were respectively cut by EcoR / XhoI and EcoRI / XhoI.T4 DNA ligase connects three fragments were constructed.Human and rat TREM1-IgGlFc/pcDNA3.1(+) recombinant plasmid were constructed,sequencing confirmed that sequence,promoter,termination of location and ORF entirely correct.Two recombinant plasmids were stably transfected into CHO cells with lipo2000 and selected with G418.RT-PCR showed that expression of the target gene was found in stably transfected CHO cells lines.The supernatants were harvested,and TREM-IgG fusion protein was purified by protocols including protein A affinity chromatography.In SDS-PAGE assay,about 66 KD bovine serum albumin and 39 KD specific protein were detected.In Western Blot assay,this product could be specifically detected by anti-human IgGl Fc antibody,which confirm it is an IgG fusion protein.
Conclusion:Construction of the Human and rats TREM1-IgGlFc fusion protein eukaryotic expression vector,and stably expressedin CHO cells.
5.Rat TREM1-IgGl fusion protein prokaryotic expression vector construction, expression and identification
Objective:To construct ratTREM1-IgGl fusion protein prokaryotic expression vector, expression and purification of recombinant rat TREM1- IgGl fusion protein.
Methods:Rat TREM1-IgGl fusion gene was cut down from rat TREM1-IgGlFc pcDNA3.1(+) recombinant plasmid by restriction enzymes,EcoRI and XhoI,and inserted into the expression vector corresponding pGEX4T-1 site,and then was transformed into E.coli BL21 where it was induced to express proteins by IPTG(isopropyl-1-thio-b Dgalactopyranoside).The expressed proteins were analyzed through SDS-PAGE,purified through Glutathione Sepharose 4B.Determine the fusion protein concentration.The expression of the fusion protein was detected by SDS-PAGE,Western Blot and PMF.
Results:Rat TREM1-IgGlFc pcDNA3.1(+) recombinant plasmid by restriction enzymes,EcoRI and XhoI.Electrophoresis confirmed that the fusion gene rat TREM1-IgGl was cut down.and inserted into the expression vector corresponding pGEX4T-1 site,and then was transformed into E.coli BL21 where it was induced to express proteins by IPTG.In SDS-PAGE assay,about 66 KD specific protein was detected. In Western Blot assay,this product could be specifically detected by anti-human IgGl Fc antibody,which confirmed it was an IgG fusion protein.17 matching peptide were measured by peptide mass fingerprint PMF,which confirmed that samples were the purpose proteins.And the concentration of the purified fusion protein was 1 mg / ml.
Conclusion:Construction rat TREM1- IgC.l fusion protein prokaryotic expression vector,expression and purification of recombinant rat TREM1- IgG.l fusion protein. Laying the foundation for further study of its as a therapeutic target molecule SAP.
6.The efficacy study of the fusion TREM1-IgG protein in the treatment of SAP rats
Objective:To observe the effect of the fusion TREM1-IgG protein in the treatment of SAP rats.
Methods:20 male SD rats were randomly divided into SAP group(group A),the control treatment(GST) group(group B) an,the fusion protein therapy group(group C) and the control group(n=5).The SAP models in rats were successfully constructed by intraperitoneal arginine injection.After six hours,the treatment was given.The GST protein and fusion protein were respectively injected in tail vein in Group B and C.The concentration was 4mg/kg.In group A,500μl saline was injected.After 24 hours,all the rats were killed.And the levels of amylase,aminopherase and creatinine in serum were detected.The histological changes were observed and scored in pancreas,liver and lung.
Results:24 hours after the treatment,there was no significant difference in the level of aminotransferase and creatinine among GST group,TREM1-IgG fusion protein group and SAP group,The level of serum amylase in TREM1-IgG fusion protein group had a downward trend.In TREM1-IgG fusion protein group,the pathology scores in pancreatic inflammation,bleeding,necrosis,lung inflammation and total organ damage had significantly improved than the other two groups(P＜0.05).
Conclusion:The fusion TREM1-IgG protein can effectively alleviate the inflammatory response and reduce damage to pancreas and lung.In SAP rats,it was proved that TREM-1 as a therapeutic target molecule in SAP is possible.
Through this study,the subjects come to the following conclusions:
1.TREM-1 involved in severe acute pancreatitis at the "excessive" inflammatory response,the expression level of TREM-1 was obviously high when the secondary infection happened.
2.The TREM-1 gene expression in the peripheral blood was a non-invasive,rapid and economic indictor in intra-abdominal secondary infection of SAP patients.The use of TREM-1 was prospected to judge the secondary infection and the time of surgery intervene.
3.Successfully constructed the eukaryotic expression vector of human TREM1-IgG fusion protein and achieved stable expression in CHO cells.
4.Successfully constructed the prokaryotic expression vector of rat TREM1-IgG fusion protein and achieved stable expression and purification.
5.The fusion TREM1-IgG protein can effectively alleviate the inflammatory response and reduce damage to pancreas and lung.In SAP rats,it was proved that TREM-1 as a therapeutic target molecule in SAP is possible.
Summary:The subject demonstrated that TREM-1 played an important role in the process of severe acute pancreatitis and had the diagnosis value in the intra-abdominal secondary infection.Construction and expression of TREM-1 / IgGl fusion protein eukaryotic and prokaryotic expression vector,through the intervention of SAP rats proved TREM-1 as a therapeutic target molecule SAP value.To laying the foundation for further study of follow-up search the natural ligand of TREM-1,and providing new ideas of SAP pathogenesis and comprehensive treatment.
引文
1 Bouchon A, Facchetti F, Weigand MA, et al. TREM-1 amplifies inflammation and is a crucial mediator of septic shock. Nature. 2001, 410:1103-1107.
2 Da-Yu Wang, Ren-Yi Qin, Zheng-Ren Liu, Manoj Kumar Gupta, Qing Chang Expression of TREM-1 mRNA in acute pancreatitis. World J Gastroenterol 2004, 10:2744-2746.
3 Gibot S, Kolopp-Sarda NW, Behe MC, et al .A soluble form of the triggering receptor expressed on myeloid cells-1 modulates the inflammatory response in murine sepsis. J Exp Med, 2004, 200:1419-1426.
4 Richeldi L, Mariani M, Losi M, et al. Triggering receptor expressed on myeloid cells: role in the diagnosis of lung infections. Eur Respir J. 2004,24(2):247-250.
5 Tzivras M, Koussoulas V, Giamarellos-Bourboulis E J, et al. Role of soluble trigering receptor expressed on myeloid cells in inflammatory bowel disease. World J Gastroenterol. 2006, 12(21):3416-3419.
6 Determann RM, Weisfeh M, de Gans J, et al .Soluble triggering receptor expressed on myeloid cells 1: a biomarker for bacterial meningitis. Intensive Care Med. 2006, 32(8):1243-1247.
7 Radaev S, Kattah M, Rostro B, et al. Crystal strueture of the human myeloid cell activating receptor TREM-1. Structure. 2003, 11:1527-1535.
8 Ramanathan B, Minton JE, Ross CR, Christopher RR, et al. Characterization of bovine cDNA encoding triggering receptor expressed on myeloid cells 1. Vet Immunopathol.2004,102(1-2):85-89.
9 Bouchon A, Dietrich J, Colonna M. Cutting edge:inflammatory responses can be triggered by TREM-1, a novel receptor expressed on neutrophils and monocytes. J Immunol. 2000,164(10):4991-4995.
10 Colonna M, Facehetti F. TREM-1: a new player in acute inflammatory responses. J Infect Dis. 2003, 187:S397-S401.
11 Washington A V, Quigley L, McVicar D W. Initial characterization of TREM-like transcript(TLT)-1:a putative inhibitory receptor within the TREM cluster. Blood. 2002,100(10):3822-3824.
12 Mohamadzadeh M, Coberley S S, Olinger G G, et al. Activation of triggering receptor expressed on myeloid cells-1 on human neutrophils by marburg and ebola viruses. J Virol. 2006, 80(14):7235-7244.
13 Murakami Y, Akahoshi T, Hayashi I, et al. Induction of triggering receptor expressed on myeloid cells 1 in murine resident peritoneal macrophages by monosedium urate monohydrate crystals. Arthritis Rheum. 2006, 54(2):455-462.
14 Bleharski JR, Kiessler V, Buonsanti C, et al. A role for triggering receptor expressed on myeloid cells-1 in host defense during the early-induced and adaptive phases of the immune response. J Immunol. 2003,170(7):3812-3818.
15 Bradley EL. A clinically based classification system for acute pancreatitis: summary of the Atlanta Symposium. Arch Surg. 1993,128(5):586-590.
16 Appelros S, Borgstrom A. Incidence, aetiology and mortality rate of acute pancreatitis over 10 years in a defined urban population in Sweden. Br J Surg. 1999 Apr, 86(4):465-70.
17 Rinderkencht H. Fatal pancreatits, a consequence of excessive leukocyte stimulation[J]. Int J Pancreatol, 1988, 3(2-3):105-112.
18 Norman J. The role of cytokines in the pathogenesis of acute pancreatitis. Am J Surg. 1998, 175(1):76-83.
19 Bhatia M, Brady M, Shokuhi S, et al. Inflammatory mediators in acute pancreatitis. J Path. 2000, 190(2):117-125.
20 Karne S, Gorelick FS. Etiopathogenesis of acute pancreatitis. Surg Clin Nor Amer. 1999, 79(4):699-710.
21 Wilson PG, Manji M, Neoptolemos JP. Acute pancreatitis as a model of sepsis. J Antimicrob Chemother. 1998,41(Suppl A):51-63.
22 Ulrich CD, Kopras E, WuY, et al. Hereditary pancreatitis:epidemiology, molecules,mutations,and models. J Lab Clin Med. 2000,136(4):260-274.
23 Imrie CW. Prognostic indicators in acute pancreatitis. Can J Gastroenterol. 2003, 17(5): 325-8.
24 Balthazar EJ, Robinson DL, Alec J,et al.Acute pancreatitis: prognostic valuel of CT in establishing prognosis. Radiology. 1990,174:331-336.
25 Balthazar EJ. CT diagnosis and staging of acute pancreatitis. Radiol Clin North Am. 1989, 27(1):19-36.
26 Mortele KJ, Mergo PJ, Taylor HM,et al. Renal and perirenal space involvement in acute pancreatitis: spiral CT findings. Abdom Imaging. 2000,25(3): 272-278.
28 Balthazar EJ, Freeny PC, van Sonnenberg E. Imaging and intervention in acute pancrestitis. Radiology. 1994,193(2): 297-306.
29 Paye F, Rotman N, Radier C, et al.Percutaneous aspiration for bacteriological studies in patients with necrotizing panereatitis.Br J Surg. 1998, 85(6):755-759.
30 Nochi H, Aoki N, Oikawa K, et al.Modulation of hepatic granulomatous responses by transgene expression of DAP12 or TREM-1-lg molecules.Am J Pathol. 2003, 162(4):1191-1201.
31 Cohen J.TREM-1 in sepsis.Lancet. 2001, 358(9284): 776-778.
32 Nathan C, Ding A.TREM-1: a new regulator of innate immunity in sepsis syndrome.Nat Med. 2001, 7(5): 530-532.
33 Kemppainen E, Sainio V, Ha apiainen R, et al.Early localization of necrosis by contrast-enhanced computed tomography can predict outcome in severe acute pancreatitis.Br J Sung. 1996, 83(7): 924-929.
34 Beger HG, Isenmann R. Surgical management of necrotizing pancreatitis. Surg Clin North Am. 1999,79(4): 783-800.
35 Saglamkaya U, Mas MR, Yasar M, et al. Penetration of meropenem and cefepim into pancreatic tissue during the course of experimental acute pancreatitis. Pancreas. 2002,24(2): 264-268.
36 Wang XP, Gong ZH, Wu K, et al.Establishment of chronic pancreatitis in rat model induced by trinitrobenzoic sulfonic acid. Chin J Pathol.2003, 32(3):267-269.
37 Yuan YF, Liu ZS, Ai ZL, et al. Effects of Chinese medicine YiBiQing on bacteria translocation in acute necrotizing pancreatitis in rabbits. Chin J Exp Surg. 2003,20(2):125-126.
38 Jerrells TR, Chapman N, Clemens DL. Animal model of alcoholic pancreatitis: role of viral infections. Pancreas. 2003,27(4):301-304.
39 Samel S, Lanig S, Lux A, et al.The gut origin of bacterial pancreatic infection during acute experimental pancreatitis in rats.Pancreatology. 2002, 2(5): 449-455.
40 Dumot JA, Conwell DL, Zuccaro GJ, et al.A randomized, double blind study of interleukin 10 for the prevention of ERCP-induced pancreatitis. Am J Gastroenterol. 2001, 96(7): 2098-2102.
41 Kruger B, Weber IA, Albrecht E, et al. Effect of hyperthermia on premature Intracellular trypsinogen activation in the exocrine pancreas. Biochem Biophys Res Commun. 2001, 282(1):159-165.
42 Ottesen LH, Bladbjerg EM, Osman M, et al. Protein C activation during the initial phase of experimental acute pancreatitis in the rabbit. Dig Surg. 1999, 16(6): 486-495.
43 Jafrary C , Yang J , Carter G, et al. Pancreatic elastase activates pulmonary nuclear factor kappa B and inhibitory kappa B, mimicking pancreatitis associated acute respiratory distress syndrome. Surgery .2000, 128 (2):225-231.
44 屠伟峰,黎介寿,朱维铭．猪急性胰腺炎与肠源性细菌/内毒素易位．中国胃肠外科杂志．2000,1(1)：22-5．
45 Lichtenstevn A, Milani R, Fernezlvain SM, et al. Acute lung injury in two experimental models of acute pancreatitis: infusion of saline orsodium taurocholate into the pancreatic duct. Crit Care Med. 2000, 28 (5): 1497-1502.
46 David C,Whitcomb.Acute Pancreatitis.The New England Journal of Medicine.2006,354:2142-50.
47 UK Working Party on Acute Pancreatitis.UK guidelines for the management of acute pancreatitis.Gut.2005,54:1-9.
48 De W aele J J,Vogelaers D,Hoste E,et al.Emergence of antibiotic resistance in infected pancreatic necrosis.Arch Surg,2004,139:1371-1375.
49 张圣道,雷若庆.当今重症急性胰腺炎治疗中的几个问题.中国实用外科杂志.2003.23(9):516.
50 赵玉沛,陈革.手术在重症急性胰腺炎治疗中的地位.中国实用外科杂志.2003,23:517.
51 Mahdy A M,Lowes D A,Galley H F,et al.Production of soluble trigering receptor expressed on myeloid cells by lipopolysaccharide-stimulated human neutrophils involves de novo protein synthesis.Clin Vaccine lmmunol.2006,13(4):492-495.
52 Gibot S,le Renard PE,Bollaert PE,Kolopp-Sarda MN,Bene MC,Faure GC,Levy B.Surface triggering receptor expressed on myeloid cells 1 expression patterns in septic shock.Intensive Care Med.2005,31:594-597.
53 钱如云,刘嘉茵.Trem-1在子宫内膜异位症中的表达.生殖与避孕.2007,27(3):182-185.
54 Gibot S,Cravoisy A,Levy B,et al.Soluble triggering receptor expressed on myeloid cells and the diagnosis of pneumonia.N Engl J Med.2004:350(5):451-458.
55 Kousseulas V,Vassiliou S,Demonakou M,et al.Soluble triggering receptor expressed on myeloid cells(strem-1):a new mediator involved in the pathogenesis of peptic ulcer disease.Eur J Gastroenterol Hepatol.2006,18(4):375-379.
56 Pugin J,Auckenthahr R,Mili N,et al.Diagnosis of ventilator associated pneumonia by bacteriologic analysis of bronchoscopic and nonbronchoseopic "blind" bronchoalveolar iavage fluid.Am Rev Respir Dis.1991,143:1121-1129.
57 王兴鹏,许国铭,袁耀宗,等.中国急性胰腺炎诊治指南(草案).中华消化杂志.2004,24:190-192.
58 Viedma JA,Perez-Mateo M,Agullo J,et al.Inflammatory response in the early prediction of severity in human acute pancreatitis.Gut.1994,35:822-827.
59 Rettally CA,Skarda S,Garza M A,et al.The usefulness of laboratory tests in the early assessment of severity of acute pancreatitis.Crit Rev Clin Lab Sci.2003,40:117-149.
60 Alfonso V,Gomez F,Lopez A,et al.Value of C-reactive protein level in the detection of necrosis in acute pancreatitis.Gastroenterol Hepatol.2003,26:288-293.
61 刘岩,路筝,李兆申.C反应蛋白与胰腺坏死的相关性探讨.中华消化杂志.2006, 26(12):829-830.
62 Janeway CA Jr, Medzbitov R. Innate inmmne recognition. Annu Rev Imnmnol. 2002,20:197-216.
63 Medzhitov R. Toll-like receptors and innate immunity. Nature Rev Immunol. 2001, 1(2):135-145.
64 Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nature Immunol. 2001,2(8):675-680.
65 Allcock R J, Barrow A D, Forbes S, et al. The human TREM gene cluster at 6p21.1 encodes both activating and inhibitory single IgV domain receptors and includes NKp44. Eur J Immunol. 2003, 33(2):7567-577.
66 Radaev S, Kattah M, Rostro B, et al. Crystal strueture of the human myeloid cell activating receptor TREM-1. Structure. 2003,11:1527-1535.
67 Matthew SK, et al. Crystal structure of human triggering receptor expressed on myeloid Cells 1 (TREM-1)at1.47A. Mol.Biol. 2004,342:1237-1248.
68 Tumbull IR, McDunn JE, Takai T, et al. DAP12 (KARAP) amplifies inflamm ation and increases mortality from endotoxemia and septic peritoritis. J Exp Med. 2005, 202(3):363-369.
69 Bouchon A, Hernandez-Munain C, Cella M, Colonna M: A DAP12-mediated pathway regulates expression of CC chemokine receptor 7 and maturation of human dendriticcells. J Exp Med. 2001, 194:1111-1122.
70 Liossis SN, Tsokos GC. Monoclonal antibodies and fusion proteins in medicine. J Allergy Clin Immunol. 2005, 116(4):721- 730.
71 Barnes LM, Bentley CM, Dickson AJ. Stability of protein production from recombinant mammalian cells. Biotechnol Bioeng. 2003, 81(6):631-9.
72 Smith D B, Johnson K S. Gene. 1988, 67: 31-40.
73 Shen Q, Li SX, Fu FH, Yuan QS, Gong Y. Two observed regions in B lymphocyte stimulator important for its biological activity. Acta Biochim Biophys Sin. 2006, 38:227-232.
74 De Leon MP, Drew AC, Glaspole IN, Suphioglu C, O'hehir RE, Rolland JM. IgE cross-reactivity between the major peanut allergen Ara h 2 and tree nut allergens. Mol Immunol. 2006, 29 in press.
75 Hughes CB, Gaber LW, Mohey el Din AB, et al. Inhibition of TNF improves survival in an experimental model of acute pancreatitis. Am Surg. 1996, 62:8-13.
76 Gukovskaya AS, Sandoval D, Zaninovic V,et al. Tumor necrosis factor regulates cell death in eaerulein-induced pancreatitis. Gastroenterology. 1996, 111 :A27729.
77 Norman J, Fink GW, Franz MG. Timing of tumor necrosis factor antagonism is critical in determining outcome in murine lethal acute pancreatitis. Surgery. 1996, 120:515-521.
78 Norman J, Franz M, Messina J, et al. Interleukin-1 receptor antagonist decreases severity of experimental acute pancreatitis. Surgery. 1995,117:648-655.
79 Denham W, Yang J, Denham D, et al. Gene targeting demonstrates additive detrimental effects of interleukin 1 and tumor necrosis factor during pancreatitis. Gastroenterology. 1997,113:1741-1747.
80 Suzuki S, Miyasaka K, Jimi A, Funakoshi A. Induction of acute pancreatitis by cerulein in human IL-6 gene transgenic mice. Pancreas. 2000 Jul, 21(1):86-92.
81 Wang D, Jin D, Wu Z, Zou W, Xu D, Zheng Z, Liu X. Therapeutic effects of human interleukin 10 gene transfer on severe acute pancreatitis in rats, an experimental study. Zhonghua Yi Xue Za Zhi. 2002 Jun 25,82(12):844-7.
82 Zou WG, Wang DS, Lang MF, Jin DY, Xu DH, Zheng ZC, Wu ZH, Liu XY.Human interleukin 10 gene therapy decreases the severity and mortality of lethal pancreatitis in rats. J Surg Res. 2002 Mar, 103(1):121-6.
83 Denham W, Fink G, Norman J, et al. Small molecule inhibition of tumor necrosis factor gene processing during acute panereatitis prevents cytokines cascade progression and attenuates pancreatitis severity. Am Surg. 1997,63:1045-1050.
84 Denham W, Yang J, Wang H, et al. Inhibition of p38 mitogen activate kinase attenuates the severity of pancreatitis-induced adult respiratory distress syndrome. Crit Care Med. 2000, 28:2567-2572.
85 Ashkenazi A, Marsters SA, Capon-DJ, et al. Protection against endotoxic shock by a tumor necrosis factor receptor immunoadhesin. Proc Natl Acad Sci USA. 1991, 88:10535-9.
86 Gater PR, Wasserman MA, Paciorek PM, et al. Inhibition of Sephadex-induced lung injury in the rat by Ro 45-2081, a tumor necrosis factor receptor fusion protein. Am J Respir Cell Mol Biol. 1996, 14:454-60.
87 Chamow SM, Duliege AM, Ammann A, et al. CD4 immunoadhesins in anti-HIV therapy: new developments. Int J Cancer Suppl. 1992,7:69-72.
88 Gibot S, Buonsanti C, Massin F, et al. Modulation of the triggering receptor expressed on the myeloid cell type 1 pathway in murine septic shock Infection and Immunity.2006, 74(5):2823-2830.
89 Colonna M: TREMs in the immune system and beyond. Nat Rev Immunol. 2003, 3:1-9.
90 Paloneva J, Manninen T, Christman G, Hovanes K, Mandelin J, Adolfsson R, Bianchin M, Bird T, Miranda R, Salmaggi A, et al. Mutations in two genes encoding different subunits of a receptor signaling complex result in an identical disease phenotype. Am J Hum Genet. 2002, 71:656-662.
91 Washington AV, Quigley L, McVicar DW. Initial characterization of TREM-like transcript (TLT)-1: A putative inhibitory receptor within the TREM cluster. Blood. 2002, 100:3822-3824.
92 Barrow AD, Astoul E, Floto A, Brooke G, Relou IA, Jennings NS, Smith KG, Ouwehand W, Famdale RW, Alexander DR, et al.Cutting edge:TREM-like transcript, a platelet immunoreceptor tyrosine-based inhibition motif encoding costimulatory immunoreceptor that enhances, rather inhibits, calcium signaling via SHP-2. J Immunol. 2004,172:5838-5842.
93 Washington AV, Schubert RL, Quigley L, Disipio T, Feltz R, Cho EH, MacVicar DW.TREM family member, TLT-1, is found exclusively in the -granules of megakaryocytes and platelets. Blood. 2004,104:1042-1047.
94 Chung DH, Seaman WE, Daws MR. Characterization of TREM-3, an activating receptor on mouse macrophages: definition of a family of single Ig-domain receptors on mouse chromosome. Eur J Immunol. 2002, 32:59-66.
95 Gibot S.TREM, new receptors mediating innate immunity. Med Sci. 2004,20:503-505.
96 McVicar DW, Taylor LS, Gosselin P, Willette-Brown J, Mikhael AI, Geahlen RL, Nakamura MC, Linnemeyer P, Seaman WE, Anderson SK, et al. DAP12-mediated signal transduction in natural killer cells. A dominant role for the Syk protein-tyrosine kinase. J Biol Chem. 1998, 273:32934-32942.
97 Radsak MP, Salih HR, Rammensee HG, Schild H. Triggering receptor expressed on myeloid cells-1 in neutrophil inflammatory responses: differential regulation of activation and survival. J Immunol. 2004, 172:4956-4963.
98 Knapp S, Gibot S, de Vos A, Versteeg HH, Colonna M,et al. Cutting edge: expression patterns of surface and soluble triggering receptor expressed on myeloid cells-1 in human endotoxinemia. J Immunol. 2004, 173:7131-7134.
99 Lucas M, Daniel L, Tomasello E, Guia S, Horschowski N, Aoki N, Figarella-Branger D, Gomez S, Vivier E. Massive inflammatory syndrome and lymphocytic immunodeficiency in KARAP/ DAP12-transgenic mice. Eur J Immunol. 2002, 32:2653-2663.
100 Gingras MC, Lapillonne H, Margolin JF. TREM-1, MDL-1, and DAP12 expression is associated with a mature stage of myeloid development. Mol Immunol. 2002, 38(11): 817-824.
101 Gibot S, le Renard PE, Bene MC, Faure GC, Bollaert PE, Levy B. Surface and soluble triggering receptor expressed on myeloid cells-1: expression patterns in murine sepsis. Crit Care Med 2005: in press.
102 Nestor Gonzalez-Roldan, Eduardo Ferat-Osorio, Rosalia Aduna-Vicente, et al. Expression of triggering receptor on myeloid cell 1 and histocompatibility complex molecules in sepsis and major abdominal surgery. World J Gastroenterol. 2005,11(47):7473-7479.
103 Marie-Claude Gingras, Helene Lapillonne, Judith F. Margolin TREM-1, MDL-1, and DAP12 expression is associated with a mature stage of myeloid development.Molecular Immunology.2001,38:817-824.
104 Tao HQ,Zhang JX,Zou SC.Clinical characteristics and management of patients with early acute severe pancreatitis:experience from a medical center in China.World J Gastroenterol.2004,10(6):919-21.
105 Kazantsev GB,Hecht DW,Rao R,Fedorak IJ,Gattuso P,Thompson K,Djuricin G,Prinz RA.Plasmid labeling confirms bacterial translocation in pancreatitis.Am J Surg.1994,167(1):201-6;discussion 206-7.
106 Reynolds JV.Gut barrier function in the surgical patient.Br J Surg.1996,83(12):1668-9.
107 吴承堂,黎沾良.急性坏死性胰腺炎并发感染的机理研究.中华外科杂志.1998,36(4):230.
108 McKay C J,Gallagher G,Brooks B,Imrie CW,Baxter JN.Increased monocyte cytokine production in association with systemic complications in acute pancreatitis.Br J Surg.1996,83(7):919-23.
109 Isenmann R,Buchler MW.Infection and acute pancreatitis.Br J Surg.1994;81(12):1707-8.
110 Sugiyama N,Akahoshi F,Kuwahara S,Kajii M,Sakaue Y,Yakumaru H,Sugiura M,Fukaya C.Synthesis and topical antiinflammatory and antiallergic activities of antioxidant o-aminophenol derivatives.J Med Chem.1994,37(13):1977-82.
111 《应用抗菌药物防治外科感染的指导意见》撰写协作组.应用抗菌药物防治外科感染的指导意见(草案).中华外科杂志.2004,42(6):374.
112 Hoerauf A,Hammer S,Muller-Myhsok B,Rupprecht H.Intra-abdominal Candida infection during acute necrotizing pancreatitis has a high prevalence and is associated with increased mortality.Crit Care Med.1998,26(12):2010-5.
113 秦帅,汤耀卿,翟洪平等.重症急性胰腺炎合并深部真菌感染易感因素剖析.外科理论与实践.2001,6(2):96-99.
114 杨雪英,李荣,陈华.急性重症胰腺炎合并真菌感染分析及对策.现代医院.2006,6(6):9.
115 Manana P,Nedelnikova K,Gurlich R.Physiology and genetics of procalcitonin.Physiol Res.2000,49 Suppl 1:S57-61.
116 Karzai W,Reinhart K.Immune modulation and sepsis.Int J Clin Pract.1997,51(4):232-7.
117 张瑾,赵诸慧.感染患儿血清降钙素原水平的变化.国外医学.儿科学分册.2000,27(1):7-9.
118 Enguix A,Rey C,Concha A,Medina A,Coto D,Dieguez MA.Comparison of procalcitonin with C-reactive protein and serum amyloid for the early diagnosis of bacterial sepsis in critically ill neonates and children.Intensive Care Med.2001,27(1):211-5.
119 李军民.血清C反应蛋白鉴别小儿急性呼吸道感染的病原体.第四军医大学学报.2002,23(13):1202.
120 Monneret G,Labaune JM,Isaac C,Bienvenu F,Putet G,Bienvenu J.Procalcitonin and C-reactive protein levels in neonatal infections.Acta Paediatr.1997,86(2):209-12.
121 Guven H,Altintop L,Baydin A,Esen S,Aygun D,Hokelek M,Doganay Z,Bek Y.Diagnostic value of procalcitonin levels as an early indicator of sepsis.Am J Emerg Med.2002,20(3):202-6.
122 Selberg O,Hecker H,Martin M,Klos A,Bautsch W,Kohl J.Discrimination of sepsis and systemic inflammatory response syndrome by determination of circulating plasma concentrations of procalcitonin,protein complement 3a,and interleukin-6.Crit Care Med.2000,28(8):2793-8.
123 Gramm HJ,Hannemann L,Reinhart K,Lode H.Sepsis:A conception in change.Possibilities and limitations of diagnosis based on clinical criteria.Dtsch Med Wochenschr.1995,120(14):498-502.
124 Gendrel D,Raymond J,Assicot M,Moulin F,Iniguez JL,Lebon P,Bohuon C.Measurement of procalcitonin levels in children with bacterial or viral meningitis.Clin Infect Dis.1997,24(6):1240-2.
125 Hatherill M,Sykes K,Mclntyre AG,Murdoch IA.Procalcitonin may help differentiate disseminated herpes simplex viral infection from bacterial sepsis in neonates.Eur J Pediatr.2000,159(3):168-9.
126 李瑞华,李晓兰,刘永娥,李振国,马荣.血清降钙素原与超敏C反应蛋白联合测定在感染性疾病中的临床意义.中国试验诊断学.2007,11(8):1105-06.
127 Bojarski K,Dabrowski A,Wallner G,Maciejewski R.Shift of zinc in serum and tissues in course of experimental acute pancreatitis.Ann Univ Mariae Curie Sklodowska.2002,57(2):74-8.
128 Shimizu T,Shiratori K,Sawada T,Kobayashi M,Hayashi N,Saotome H,Keith JC.Recombinant human interleukin-11 decreases severity of acute necrotizing pancreatitis in mice.Pancreas.2000,21(2):134-40.
129 Rongione AJ,Kusske AM,Kwan K,Ashley SW,Reber HA,McFadden DW.Interleukin 10reduces the severity of acute pancreatitis in rats.Gastroenterology.1997,112(3):960-7.
130 Van Laethem JL,Eskinazi R,Louis H,Rickaert F,Robberecht P,Deviere J.Multisystemic production of interleukin 10 limits the severity of acute pancreatitis in mice.Gut.1998,43(3):408-13.
131 Bierbach B,Meier M,Kasper-Konig W,Heimann A,Alessandri B,Horstick G,Oelert H,Kempski O.Emboli formation rather than inflammatory mediators are responsible for increased cerebral water content after conventional and assisted beating-heart myocardial revascularization in a porcine model.Stroke.2008,39(1):213-219.
132 Uhl W,Isenmann R,Buchler MW.Infections complicating pancreatitis:diagnosing,treating,preventing.New Horiz.1998,6(2 Suppl).
133 Kotani J, Usami M, Nomura H, Iso A, Kasahara H, Kuroda Y, Oyanagi H, Saitoh Y. Enteral nutrition prevents bacterial translocation but does not improve survival during acute pancreatitis. Arch Surg. 1999, 134(3):287-92.
134 Qin HL, Su ZD, Hu LG, Ding ZX, Lin QT. Effect of early intrajejunal nutrition on pancreatic pathological features and gut barrier function in dogs with acute pancreatitis. Clin Nutr. 2002 Dec, 21(6): 469-73.
135 Foitzik T, Kruschewski M, Kroesen AJ, Hotz HG, Eibl G, Buhr HJ. Does glutamine reduce bacterial translocation? A study in two animal models with impaired gut barrier. Int J Colorectal Dis. 1999, 14(3):143-9.
136 Sahin M, Yol S, Ciftci E, Baykan M, Ozer S, Akoz M, Yilmaz O, Kuru C. Does large-bowel enema reduce septic complications in acute pancreatitis? Am J Surg. 1998, 176(4):331-4.
137 Chen X, Valente JF, Alexander JW. The effect of sennosides on bacterial translocation and survival in a model of acute hemorrhagic pancreatitis. Pancreas. 1999, 18(1):39-46.
138 Gong Z, Wu Y, Wang Y, Wang C, Hou Z, Jiang Y, Jiang W, Wu X, Wang C, Xian H. Phase-compensation experiment with a 37-element adaptive optics system. 1998, 37(21):4549-52.
139 Bruns CJ, Harbison MT, Davis DW, Portera CA, Tsan R, McConkey DJ, Evans DB, Abbruzzese JL, Hicklin DJ, Radinsky R. Epidermal growth factor receptor blockade with C225 plus gemcitabine results in regression of human pancreatic carcinoma growing orthotopically in nude mice by antiangiogenic mechanisms. Clin Cancer Res. 2000, 6(5): 1936-48.
140 Kouris GJ, Liu Q, Rossi H, Djuricin G, Gattuso P, Nathan C, Weinstein RA, Prinz RA. The effect of glucagon-like peptide 2 on intestinal permeability and bacterial translocation in acute necrotizing pancreatitis. Am J Surg. 2001, 181(6):571-5.
141 Liu Q, Djuricin G, Nathan C, Gattuso P, Weinstein RA, Prinz RA. The effect of epidermal growth factor on the septic complications of acute pancreatitis. J Surg Res. 1997, 69(1): 171-7.
142 Takagi K, Isaji S. Therapeutic efficacy of continuous arterial infusion of an antibiotic and a protease inhibitor via the superior mesenteric artery for acute pancreatitis in an animal model. Pancreas. 2000,21(3):279-89.
143 Gloor B, Schmidtmann AB, Worni M, Ahmed Z, Uhl W, Buchler MW. Pancreatic sepsis:prevention and therapy. Best Pract Res Clin Gastroenterol. 2002, 16(3): 379-90.
2 Da-Yu Wang, Ren-Yi Qin, Zheng-Ren Liu, Manoj Kumar Gupta, Qing Chang Expression of TREM-1 mRNA in acute pancreatitis. World J Gastroenterol 2004, 10:2744-2746.
3 Gibot S, Kolopp-Sarda NW, Behe MC, et al .A soluble form of the triggering receptor expressed on myeloid cells-1 modulates the inflammatory response in murine sepsis. J Exp Med, 2004, 200:1419-1426.
4 Richeldi L, Mariani M, Losi M, et al. Triggering receptor expressed on myeloid cells: role in the diagnosis of lung infections. Eur Respir J. 2004,24(2):247-250.
5 Tzivras M, Koussoulas V, Giamarellos-Bourboulis E J, et al. Role of soluble trigering receptor expressed on myeloid cells in inflammatory bowel disease. World J Gastroenterol. 2006, 12(21):3416-3419.
6 Determann RM, Weisfeh M, de Gans J, et al .Soluble triggering receptor expressed on myeloid cells 1: a biomarker for bacterial meningitis. Intensive Care Med. 2006, 32(8):1243-1247.
7 Radaev S, Kattah M, Rostro B, et al. Crystal strueture of the human myeloid cell activating receptor TREM-1. Structure. 2003, 11:1527-1535.
8 Ramanathan B, Minton JE, Ross CR, Christopher RR, et al. Characterization of bovine cDNA encoding triggering receptor expressed on myeloid cells 1. Vet Immunopathol.2004,102(1-2):85-89.
9 Bouchon A, Dietrich J, Colonna M. Cutting edge:inflammatory responses can be triggered by TREM-1, a novel receptor expressed on neutrophils and monocytes. J Immunol. 2000,164(10):4991-4995.
10 Colonna M, Facehetti F. TREM-1: a new player in acute inflammatory responses. J Infect Dis. 2003, 187:S397-S401.
11 Washington A V, Quigley L, McVicar D W. Initial characterization of TREM-like transcript(TLT)-1:a putative inhibitory receptor within the TREM cluster. Blood. 2002,100(10):3822-3824.
12 Mohamadzadeh M, Coberley S S, Olinger G G, et al. Activation of triggering receptor expressed on myeloid cells-1 on human neutrophils by marburg and ebola viruses. J Virol. 2006, 80(14):7235-7244.
13 Murakami Y, Akahoshi T, Hayashi I, et al. Induction of triggering receptor expressed on myeloid cells 1 in murine resident peritoneal macrophages by monosedium urate monohydrate crystals. Arthritis Rheum. 2006, 54(2):455-462.
14 Bleharski JR, Kiessler V, Buonsanti C, et al. A role for triggering receptor expressed on myeloid cells-1 in host defense during the early-induced and adaptive phases of the immune response. J Immunol. 2003,170(7):3812-3818.
15 Bradley EL. A clinically based classification system for acute pancreatitis: summary of the Atlanta Symposium. Arch Surg. 1993,128(5):586-590.
16 Appelros S, Borgstrom A. Incidence, aetiology and mortality rate of acute pancreatitis over 10 years in a defined urban population in Sweden. Br J Surg. 1999 Apr, 86(4):465-70.
17 Rinderkencht H. Fatal pancreatits, a consequence of excessive leukocyte stimulation[J]. Int J Pancreatol, 1988, 3(2-3):105-112.
18 Norman J. The role of cytokines in the pathogenesis of acute pancreatitis. Am J Surg. 1998, 175(1):76-83.
19 Bhatia M, Brady M, Shokuhi S, et al. Inflammatory mediators in acute pancreatitis. J Path. 2000, 190(2):117-125.
20 Karne S, Gorelick FS. Etiopathogenesis of acute pancreatitis. Surg Clin Nor Amer. 1999, 79(4):699-710.
21 Wilson PG, Manji M, Neoptolemos JP. Acute pancreatitis as a model of sepsis. J Antimicrob Chemother. 1998,41(Suppl A):51-63.
22 Ulrich CD, Kopras E, WuY, et al. Hereditary pancreatitis:epidemiology, molecules,mutations,and models. J Lab Clin Med. 2000,136(4):260-274.
23 Imrie CW. Prognostic indicators in acute pancreatitis. Can J Gastroenterol. 2003, 17(5): 325-8.
24 Balthazar EJ, Robinson DL, Alec J,et al.Acute pancreatitis: prognostic valuel of CT in establishing prognosis. Radiology. 1990,174:331-336.
25 Balthazar EJ. CT diagnosis and staging of acute pancreatitis. Radiol Clin North Am. 1989, 27(1):19-36.
26 Mortele KJ, Mergo PJ, Taylor HM,et al. Renal and perirenal space involvement in acute pancreatitis: spiral CT findings. Abdom Imaging. 2000,25(3): 272-278.
28 Balthazar EJ, Freeny PC, van Sonnenberg E. Imaging and intervention in acute pancrestitis. Radiology. 1994,193(2): 297-306.
29 Paye F, Rotman N, Radier C, et al.Percutaneous aspiration for bacteriological studies in patients with necrotizing panereatitis.Br J Surg. 1998, 85(6):755-759.
30 Nochi H, Aoki N, Oikawa K, et al.Modulation of hepatic granulomatous responses by transgene expression of DAP12 or TREM-1-lg molecules.Am J Pathol. 2003, 162(4):1191-1201.
31 Cohen J.TREM-1 in sepsis.Lancet. 2001, 358(9284): 776-778.
32 Nathan C, Ding A.TREM-1: a new regulator of innate immunity in sepsis syndrome.Nat Med. 2001, 7(5): 530-532.
33 Kemppainen E, Sainio V, Ha apiainen R, et al.Early localization of necrosis by contrast-enhanced computed tomography can predict outcome in severe acute pancreatitis.Br J Sung. 1996, 83(7): 924-929.
34 Beger HG, Isenmann R. Surgical management of necrotizing pancreatitis. Surg Clin North Am. 1999,79(4): 783-800.
35 Saglamkaya U, Mas MR, Yasar M, et al. Penetration of meropenem and cefepim into pancreatic tissue during the course of experimental acute pancreatitis. Pancreas. 2002,24(2): 264-268.
36 Wang XP, Gong ZH, Wu K, et al.Establishment of chronic pancreatitis in rat model induced by trinitrobenzoic sulfonic acid. Chin J Pathol.2003, 32(3):267-269.
37 Yuan YF, Liu ZS, Ai ZL, et al. Effects of Chinese medicine YiBiQing on bacteria translocation in acute necrotizing pancreatitis in rabbits. Chin J Exp Surg. 2003,20(2):125-126.
38 Jerrells TR, Chapman N, Clemens DL. Animal model of alcoholic pancreatitis: role of viral infections. Pancreas. 2003,27(4):301-304.
39 Samel S, Lanig S, Lux A, et al.The gut origin of bacterial pancreatic infection during acute experimental pancreatitis in rats.Pancreatology. 2002, 2(5): 449-455.
40 Dumot JA, Conwell DL, Zuccaro GJ, et al.A randomized, double blind study of interleukin 10 for the prevention of ERCP-induced pancreatitis. Am J Gastroenterol. 2001, 96(7): 2098-2102.
41 Kruger B, Weber IA, Albrecht E, et al. Effect of hyperthermia on premature Intracellular trypsinogen activation in the exocrine pancreas. Biochem Biophys Res Commun. 2001, 282(1):159-165.
42 Ottesen LH, Bladbjerg EM, Osman M, et al. Protein C activation during the initial phase of experimental acute pancreatitis in the rabbit. Dig Surg. 1999, 16(6): 486-495.
43 Jafrary C , Yang J , Carter G, et al. Pancreatic elastase activates pulmonary nuclear factor kappa B and inhibitory kappa B, mimicking pancreatitis associated acute respiratory distress syndrome. Surgery .2000, 128 (2):225-231.
44 屠伟峰,黎介寿,朱维铭．猪急性胰腺炎与肠源性细菌/内毒素易位．中国胃肠外科杂志．2000,1(1)：22-5．
45 Lichtenstevn A, Milani R, Fernezlvain SM, et al. Acute lung injury in two experimental models of acute pancreatitis: infusion of saline orsodium taurocholate into the pancreatic duct. Crit Care Med. 2000, 28 (5): 1497-1502.
46 David C,Whitcomb.Acute Pancreatitis.The New England Journal of Medicine.2006,354:2142-50.
47 UK Working Party on Acute Pancreatitis.UK guidelines for the management of acute pancreatitis.Gut.2005,54:1-9.
48 De W aele J J,Vogelaers D,Hoste E,et al.Emergence of antibiotic resistance in infected pancreatic necrosis.Arch Surg,2004,139:1371-1375.
49 张圣道,雷若庆.当今重症急性胰腺炎治疗中的几个问题.中国实用外科杂志.2003.23(9):516.
50 赵玉沛,陈革.手术在重症急性胰腺炎治疗中的地位.中国实用外科杂志.2003,23:517.
51 Mahdy A M,Lowes D A,Galley H F,et al.Production of soluble trigering receptor expressed on myeloid cells by lipopolysaccharide-stimulated human neutrophils involves de novo protein synthesis.Clin Vaccine lmmunol.2006,13(4):492-495.
52 Gibot S,le Renard PE,Bollaert PE,Kolopp-Sarda MN,Bene MC,Faure GC,Levy B.Surface triggering receptor expressed on myeloid cells 1 expression patterns in septic shock.Intensive Care Med.2005,31:594-597.
53 钱如云,刘嘉茵.Trem-1在子宫内膜异位症中的表达.生殖与避孕.2007,27(3):182-185.
54 Gibot S,Cravoisy A,Levy B,et al.Soluble triggering receptor expressed on myeloid cells and the diagnosis of pneumonia.N Engl J Med.2004:350(5):451-458.
55 Kousseulas V,Vassiliou S,Demonakou M,et al.Soluble triggering receptor expressed on myeloid cells(strem-1):a new mediator involved in the pathogenesis of peptic ulcer disease.Eur J Gastroenterol Hepatol.2006,18(4):375-379.
56 Pugin J,Auckenthahr R,Mili N,et al.Diagnosis of ventilator associated pneumonia by bacteriologic analysis of bronchoscopic and nonbronchoseopic "blind" bronchoalveolar iavage fluid.Am Rev Respir Dis.1991,143:1121-1129.
57 王兴鹏,许国铭,袁耀宗,等.中国急性胰腺炎诊治指南(草案).中华消化杂志.2004,24:190-192.
58 Viedma JA,Perez-Mateo M,Agullo J,et al.Inflammatory response in the early prediction of severity in human acute pancreatitis.Gut.1994,35:822-827.
59 Rettally CA,Skarda S,Garza M A,et al.The usefulness of laboratory tests in the early assessment of severity of acute pancreatitis.Crit Rev Clin Lab Sci.2003,40:117-149.
60 Alfonso V,Gomez F,Lopez A,et al.Value of C-reactive protein level in the detection of necrosis in acute pancreatitis.Gastroenterol Hepatol.2003,26:288-293.
61 刘岩,路筝,李兆申.C反应蛋白与胰腺坏死的相关性探讨.中华消化杂志.2006, 26(12):829-830.
62 Janeway CA Jr, Medzbitov R. Innate inmmne recognition. Annu Rev Imnmnol. 2002,20:197-216.
63 Medzhitov R. Toll-like receptors and innate immunity. Nature Rev Immunol. 2001, 1(2):135-145.
64 Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nature Immunol. 2001,2(8):675-680.
65 Allcock R J, Barrow A D, Forbes S, et al. The human TREM gene cluster at 6p21.1 encodes both activating and inhibitory single IgV domain receptors and includes NKp44. Eur J Immunol. 2003, 33(2):7567-577.
66 Radaev S, Kattah M, Rostro B, et al. Crystal strueture of the human myeloid cell activating receptor TREM-1. Structure. 2003,11:1527-1535.
67 Matthew SK, et al. Crystal structure of human triggering receptor expressed on myeloid Cells 1 (TREM-1)at1.47A. Mol.Biol. 2004,342:1237-1248.
68 Tumbull IR, McDunn JE, Takai T, et al. DAP12 (KARAP) amplifies inflamm ation and increases mortality from endotoxemia and septic peritoritis. J Exp Med. 2005, 202(3):363-369.
69 Bouchon A, Hernandez-Munain C, Cella M, Colonna M: A DAP12-mediated pathway regulates expression of CC chemokine receptor 7 and maturation of human dendriticcells. J Exp Med. 2001, 194:1111-1122.
70 Liossis SN, Tsokos GC. Monoclonal antibodies and fusion proteins in medicine. J Allergy Clin Immunol. 2005, 116(4):721- 730.
71 Barnes LM, Bentley CM, Dickson AJ. Stability of protein production from recombinant mammalian cells. Biotechnol Bioeng. 2003, 81(6):631-9.
72 Smith D B, Johnson K S. Gene. 1988, 67: 31-40.
73 Shen Q, Li SX, Fu FH, Yuan QS, Gong Y. Two observed regions in B lymphocyte stimulator important for its biological activity. Acta Biochim Biophys Sin. 2006, 38:227-232.
74 De Leon MP, Drew AC, Glaspole IN, Suphioglu C, O'hehir RE, Rolland JM. IgE cross-reactivity between the major peanut allergen Ara h 2 and tree nut allergens. Mol Immunol. 2006, 29 in press.
75 Hughes CB, Gaber LW, Mohey el Din AB, et al. Inhibition of TNF improves survival in an experimental model of acute pancreatitis. Am Surg. 1996, 62:8-13.
76 Gukovskaya AS, Sandoval D, Zaninovic V,et al. Tumor necrosis factor regulates cell death in eaerulein-induced pancreatitis. Gastroenterology. 1996, 111 :A27729.
77 Norman J, Fink GW, Franz MG. Timing of tumor necrosis factor antagonism is critical in determining outcome in murine lethal acute pancreatitis. Surgery. 1996, 120:515-521.
78 Norman J, Franz M, Messina J, et al. Interleukin-1 receptor antagonist decreases severity of experimental acute pancreatitis. Surgery. 1995,117:648-655.
79 Denham W, Yang J, Denham D, et al. Gene targeting demonstrates additive detrimental effects of interleukin 1 and tumor necrosis factor during pancreatitis. Gastroenterology. 1997,113:1741-1747.
80 Suzuki S, Miyasaka K, Jimi A, Funakoshi A. Induction of acute pancreatitis by cerulein in human IL-6 gene transgenic mice. Pancreas. 2000 Jul, 21(1):86-92.
81 Wang D, Jin D, Wu Z, Zou W, Xu D, Zheng Z, Liu X. Therapeutic effects of human interleukin 10 gene transfer on severe acute pancreatitis in rats, an experimental study. Zhonghua Yi Xue Za Zhi. 2002 Jun 25,82(12):844-7.
82 Zou WG, Wang DS, Lang MF, Jin DY, Xu DH, Zheng ZC, Wu ZH, Liu XY.Human interleukin 10 gene therapy decreases the severity and mortality of lethal pancreatitis in rats. J Surg Res. 2002 Mar, 103(1):121-6.
83 Denham W, Fink G, Norman J, et al. Small molecule inhibition of tumor necrosis factor gene processing during acute panereatitis prevents cytokines cascade progression and attenuates pancreatitis severity. Am Surg. 1997,63:1045-1050.
84 Denham W, Yang J, Wang H, et al. Inhibition of p38 mitogen activate kinase attenuates the severity of pancreatitis-induced adult respiratory distress syndrome. Crit Care Med. 2000, 28:2567-2572.
85 Ashkenazi A, Marsters SA, Capon-DJ, et al. Protection against endotoxic shock by a tumor necrosis factor receptor immunoadhesin. Proc Natl Acad Sci USA. 1991, 88:10535-9.
86 Gater PR, Wasserman MA, Paciorek PM, et al. Inhibition of Sephadex-induced lung injury in the rat by Ro 45-2081, a tumor necrosis factor receptor fusion protein. Am J Respir Cell Mol Biol. 1996, 14:454-60.
87 Chamow SM, Duliege AM, Ammann A, et al. CD4 immunoadhesins in anti-HIV therapy: new developments. Int J Cancer Suppl. 1992,7:69-72.
88 Gibot S, Buonsanti C, Massin F, et al. Modulation of the triggering receptor expressed on the myeloid cell type 1 pathway in murine septic shock Infection and Immunity.2006, 74(5):2823-2830.
89 Colonna M: TREMs in the immune system and beyond. Nat Rev Immunol. 2003, 3:1-9.
90 Paloneva J, Manninen T, Christman G, Hovanes K, Mandelin J, Adolfsson R, Bianchin M, Bird T, Miranda R, Salmaggi A, et al. Mutations in two genes encoding different subunits of a receptor signaling complex result in an identical disease phenotype. Am J Hum Genet. 2002, 71:656-662.
91 Washington AV, Quigley L, McVicar DW. Initial characterization of TREM-like transcript (TLT)-1: A putative inhibitory receptor within the TREM cluster. Blood. 2002, 100:3822-3824.
92 Barrow AD, Astoul E, Floto A, Brooke G, Relou IA, Jennings NS, Smith KG, Ouwehand W, Famdale RW, Alexander DR, et al.Cutting edge:TREM-like transcript, a platelet immunoreceptor tyrosine-based inhibition motif encoding costimulatory immunoreceptor that enhances, rather inhibits, calcium signaling via SHP-2. J Immunol. 2004,172:5838-5842.
93 Washington AV, Schubert RL, Quigley L, Disipio T, Feltz R, Cho EH, MacVicar DW.TREM family member, TLT-1, is found exclusively in the -granules of megakaryocytes and platelets. Blood. 2004,104:1042-1047.
94 Chung DH, Seaman WE, Daws MR. Characterization of TREM-3, an activating receptor on mouse macrophages: definition of a family of single Ig-domain receptors on mouse chromosome. Eur J Immunol. 2002, 32:59-66.
95 Gibot S.TREM, new receptors mediating innate immunity. Med Sci. 2004,20:503-505.
96 McVicar DW, Taylor LS, Gosselin P, Willette-Brown J, Mikhael AI, Geahlen RL, Nakamura MC, Linnemeyer P, Seaman WE, Anderson SK, et al. DAP12-mediated signal transduction in natural killer cells. A dominant role for the Syk protein-tyrosine kinase. J Biol Chem. 1998, 273:32934-32942.
97 Radsak MP, Salih HR, Rammensee HG, Schild H. Triggering receptor expressed on myeloid cells-1 in neutrophil inflammatory responses: differential regulation of activation and survival. J Immunol. 2004, 172:4956-4963.
98 Knapp S, Gibot S, de Vos A, Versteeg HH, Colonna M,et al. Cutting edge: expression patterns of surface and soluble triggering receptor expressed on myeloid cells-1 in human endotoxinemia. J Immunol. 2004, 173:7131-7134.
99 Lucas M, Daniel L, Tomasello E, Guia S, Horschowski N, Aoki N, Figarella-Branger D, Gomez S, Vivier E. Massive inflammatory syndrome and lymphocytic immunodeficiency in KARAP/ DAP12-transgenic mice. Eur J Immunol. 2002, 32:2653-2663.
100 Gingras MC, Lapillonne H, Margolin JF. TREM-1, MDL-1, and DAP12 expression is associated with a mature stage of myeloid development. Mol Immunol. 2002, 38(11): 817-824.
101 Gibot S, le Renard PE, Bene MC, Faure GC, Bollaert PE, Levy B. Surface and soluble triggering receptor expressed on myeloid cells-1: expression patterns in murine sepsis. Crit Care Med 2005: in press.
102 Nestor Gonzalez-Roldan, Eduardo Ferat-Osorio, Rosalia Aduna-Vicente, et al. Expression of triggering receptor on myeloid cell 1 and histocompatibility complex molecules in sepsis and major abdominal surgery. World J Gastroenterol. 2005,11(47):7473-7479.
103 Marie-Claude Gingras, Helene Lapillonne, Judith F. Margolin TREM-1, MDL-1, and DAP12 expression is associated with a mature stage of myeloid development.Molecular Immunology.2001,38:817-824.
104 Tao HQ,Zhang JX,Zou SC.Clinical characteristics and management of patients with early acute severe pancreatitis:experience from a medical center in China.World J Gastroenterol.2004,10(6):919-21.
105 Kazantsev GB,Hecht DW,Rao R,Fedorak IJ,Gattuso P,Thompson K,Djuricin G,Prinz RA.Plasmid labeling confirms bacterial translocation in pancreatitis.Am J Surg.1994,167(1):201-6;discussion 206-7.
106 Reynolds JV.Gut barrier function in the surgical patient.Br J Surg.1996,83(12):1668-9.
107 吴承堂,黎沾良.急性坏死性胰腺炎并发感染的机理研究.中华外科杂志.1998,36(4):230.
108 McKay C J,Gallagher G,Brooks B,Imrie CW,Baxter JN.Increased monocyte cytokine production in association with systemic complications in acute pancreatitis.Br J Surg.1996,83(7):919-23.
109 Isenmann R,Buchler MW.Infection and acute pancreatitis.Br J Surg.1994;81(12):1707-8.
110 Sugiyama N,Akahoshi F,Kuwahara S,Kajii M,Sakaue Y,Yakumaru H,Sugiura M,Fukaya C.Synthesis and topical antiinflammatory and antiallergic activities of antioxidant o-aminophenol derivatives.J Med Chem.1994,37(13):1977-82.
111 《应用抗菌药物防治外科感染的指导意见》撰写协作组.应用抗菌药物防治外科感染的指导意见(草案).中华外科杂志.2004,42(6):374.
112 Hoerauf A,Hammer S,Muller-Myhsok B,Rupprecht H.Intra-abdominal Candida infection during acute necrotizing pancreatitis has a high prevalence and is associated with increased mortality.Crit Care Med.1998,26(12):2010-5.
113 秦帅,汤耀卿,翟洪平等.重症急性胰腺炎合并深部真菌感染易感因素剖析.外科理论与实践.2001,6(2):96-99.
114 杨雪英,李荣,陈华.急性重症胰腺炎合并真菌感染分析及对策.现代医院.2006,6(6):9.
115 Manana P,Nedelnikova K,Gurlich R.Physiology and genetics of procalcitonin.Physiol Res.2000,49 Suppl 1:S57-61.
116 Karzai W,Reinhart K.Immune modulation and sepsis.Int J Clin Pract.1997,51(4):232-7.
117 张瑾,赵诸慧.感染患儿血清降钙素原水平的变化.国外医学.儿科学分册.2000,27(1):7-9.
118 Enguix A,Rey C,Concha A,Medina A,Coto D,Dieguez MA.Comparison of procalcitonin with C-reactive protein and serum amyloid for the early diagnosis of bacterial sepsis in critically ill neonates and children.Intensive Care Med.2001,27(1):211-5.
119 李军民.血清C反应蛋白鉴别小儿急性呼吸道感染的病原体.第四军医大学学报.2002,23(13):1202.
120 Monneret G,Labaune JM,Isaac C,Bienvenu F,Putet G,Bienvenu J.Procalcitonin and C-reactive protein levels in neonatal infections.Acta Paediatr.1997,86(2):209-12.
121 Guven H,Altintop L,Baydin A,Esen S,Aygun D,Hokelek M,Doganay Z,Bek Y.Diagnostic value of procalcitonin levels as an early indicator of sepsis.Am J Emerg Med.2002,20(3):202-6.
122 Selberg O,Hecker H,Martin M,Klos A,Bautsch W,Kohl J.Discrimination of sepsis and systemic inflammatory response syndrome by determination of circulating plasma concentrations of procalcitonin,protein complement 3a,and interleukin-6.Crit Care Med.2000,28(8):2793-8.
123 Gramm HJ,Hannemann L,Reinhart K,Lode H.Sepsis:A conception in change.Possibilities and limitations of diagnosis based on clinical criteria.Dtsch Med Wochenschr.1995,120(14):498-502.
124 Gendrel D,Raymond J,Assicot M,Moulin F,Iniguez JL,Lebon P,Bohuon C.Measurement of procalcitonin levels in children with bacterial or viral meningitis.Clin Infect Dis.1997,24(6):1240-2.
125 Hatherill M,Sykes K,Mclntyre AG,Murdoch IA.Procalcitonin may help differentiate disseminated herpes simplex viral infection from bacterial sepsis in neonates.Eur J Pediatr.2000,159(3):168-9.
126 李瑞华,李晓兰,刘永娥,李振国,马荣.血清降钙素原与超敏C反应蛋白联合测定在感染性疾病中的临床意义.中国试验诊断学.2007,11(8):1105-06.
127 Bojarski K,Dabrowski A,Wallner G,Maciejewski R.Shift of zinc in serum and tissues in course of experimental acute pancreatitis.Ann Univ Mariae Curie Sklodowska.2002,57(2):74-8.
128 Shimizu T,Shiratori K,Sawada T,Kobayashi M,Hayashi N,Saotome H,Keith JC.Recombinant human interleukin-11 decreases severity of acute necrotizing pancreatitis in mice.Pancreas.2000,21(2):134-40.
129 Rongione AJ,Kusske AM,Kwan K,Ashley SW,Reber HA,McFadden DW.Interleukin 10reduces the severity of acute pancreatitis in rats.Gastroenterology.1997,112(3):960-7.
130 Van Laethem JL,Eskinazi R,Louis H,Rickaert F,Robberecht P,Deviere J.Multisystemic production of interleukin 10 limits the severity of acute pancreatitis in mice.Gut.1998,43(3):408-13.
131 Bierbach B,Meier M,Kasper-Konig W,Heimann A,Alessandri B,Horstick G,Oelert H,Kempski O.Emboli formation rather than inflammatory mediators are responsible for increased cerebral water content after conventional and assisted beating-heart myocardial revascularization in a porcine model.Stroke.2008,39(1):213-219.
132 Uhl W,Isenmann R,Buchler MW.Infections complicating pancreatitis:diagnosing,treating,preventing.New Horiz.1998,6(2 Suppl).
133 Kotani J, Usami M, Nomura H, Iso A, Kasahara H, Kuroda Y, Oyanagi H, Saitoh Y. Enteral nutrition prevents bacterial translocation but does not improve survival during acute pancreatitis. Arch Surg. 1999, 134(3):287-92.
134 Qin HL, Su ZD, Hu LG, Ding ZX, Lin QT. Effect of early intrajejunal nutrition on pancreatic pathological features and gut barrier function in dogs with acute pancreatitis. Clin Nutr. 2002 Dec, 21(6): 469-73.
135 Foitzik T, Kruschewski M, Kroesen AJ, Hotz HG, Eibl G, Buhr HJ. Does glutamine reduce bacterial translocation? A study in two animal models with impaired gut barrier. Int J Colorectal Dis. 1999, 14(3):143-9.
136 Sahin M, Yol S, Ciftci E, Baykan M, Ozer S, Akoz M, Yilmaz O, Kuru C. Does large-bowel enema reduce septic complications in acute pancreatitis? Am J Surg. 1998, 176(4):331-4.
137 Chen X, Valente JF, Alexander JW. The effect of sennosides on bacterial translocation and survival in a model of acute hemorrhagic pancreatitis. Pancreas. 1999, 18(1):39-46.
138 Gong Z, Wu Y, Wang Y, Wang C, Hou Z, Jiang Y, Jiang W, Wu X, Wang C, Xian H. Phase-compensation experiment with a 37-element adaptive optics system. 1998, 37(21):4549-52.
139 Bruns CJ, Harbison MT, Davis DW, Portera CA, Tsan R, McConkey DJ, Evans DB, Abbruzzese JL, Hicklin DJ, Radinsky R. Epidermal growth factor receptor blockade with C225 plus gemcitabine results in regression of human pancreatic carcinoma growing orthotopically in nude mice by antiangiogenic mechanisms. Clin Cancer Res. 2000, 6(5): 1936-48.
140 Kouris GJ, Liu Q, Rossi H, Djuricin G, Gattuso P, Nathan C, Weinstein RA, Prinz RA. The effect of glucagon-like peptide 2 on intestinal permeability and bacterial translocation in acute necrotizing pancreatitis. Am J Surg. 2001, 181(6):571-5.
141 Liu Q, Djuricin G, Nathan C, Gattuso P, Weinstein RA, Prinz RA. The effect of epidermal growth factor on the septic complications of acute pancreatitis. J Surg Res. 1997, 69(1): 171-7.
142 Takagi K, Isaji S. Therapeutic efficacy of continuous arterial infusion of an antibiotic and a protease inhibitor via the superior mesenteric artery for acute pancreatitis in an animal model. Pancreas. 2000,21(3):279-89.
143 Gloor B, Schmidtmann AB, Worni M, Ahmed Z, Uhl W, Buchler MW. Pancreatic sepsis:prevention and therapy. Best Pract Res Clin Gastroenterol. 2002, 16(3): 379-90.
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