乌司他丁对双下肢缺血再灌注后心肌损伤的保护作用
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摘要
肢体缺血再灌注损伤是临床常见的病理过程，良好的血液循环是组织细胞获得充足的氧供和营养物质并排出代谢产物的基本保证，各种原因造成的组织血液灌流量减少可使细胞发生缺血性损伤，而缺血组织器官恢复血液灌注则可能发生再灌注损伤，此连续的病理性损伤过程称为缺血再灌注损伤(ischemiareperfusion injury，IRI)，缺血再灌注损伤往往会加重组织细胞功能代谢障碍及结构破坏。随着外科技术、重症监护技术和基础医学的日益进步，缺血再灌注损伤所造成的远离器官(remote organ)损伤，即非缺血区域组织器官损伤的现象日益引起人们的关注。肢体缺血再灌注(limb ischemic perfusion，LIR)可引起远隔器官的损伤，其中心脏是最易受累的器官之一。研究认为，LIR不仅影响缺血组织的成活和功能，而且还可以累及远隔器官，严重时引发多器官功能衰竭而导致患者死亡。较为常见的是下肢缺血再灌注损伤可导致心、肺、肾和血液系统的组织及功能的受损，其中心肺是最重要的受累靶器官，临床表现为心律失常、血压下降、肺水肿甚至呼吸和循环功能衰竭等。因此研究IR对远隔部位组织器官的脏器保护有积极的临床意义。
大量研究结果显示，肢体IR损伤与自由基增多有关，肢体缺血再灌注后产生大量氧自由基(OFR)，在损伤肢体的同时，自由基可经其远端效应或直接激活多聚核中性粒细胞(PMN)，使远隔组织器官遭受损伤。PMN活化引发的“呼吸爆发(respiratory burst)”是造成远隔器官损伤的另一原因，激活的PMN相互聚集堵塞微血管，损伤血管内皮细胞，产生并释放大量自由基。实验表明，再灌注后5～30min呼吸爆发氧自由基释放达到高峰。止血带机械性加压致肢体缺血、手术创伤、组织受到严重损害，激发体内多种渠道产生氧自由基，特别是肌肉组织低灌注后缺血组织中通过黄嘌呤-黄嘌呤氧化酶机制，更是产生氧自由基的主要来源。再灌注损伤发生时，出于酶大量进入血液内，故血中酶含量的变化可作为缺血再灌注损伤中肌细胞损害最敏感的一项指标。骨骼肌缺血后的再灌注期产生大量的氧自由基，机体通过抗氧化酶清除氧自由基，而起到保护骨骼肌组织的作用。肢体缺血再灌注是炎症连锁反应的诱发因素，再灌注后可激活中性粒细胞，产生大量氧自由基及炎性介质，使机体处于全身炎症反应状态，引起远隔多器官损伤，除了外科缺血因素以外，炎性因子的水平与心肌损伤之间也是密切相关的，炎症反应越重，心肌损伤也越重，即炎症反应会加重心肌损伤的程度。
乌司他丁(ulinastatin，UTI)是从健康成年男性尿液中提取的广谱蛋白酶抑制剂，是一种糖蛋白，由143个氨基酸组成，相对分子量约为67 000。目前其一级结构尚不清楚，但确定了同类胰蛋白酶抑制剂的一级结构。其基本骨架由两个所谓的Kunitz(库纳斯)型区域组成，具有两个活性功能区，此两个活性区均有很广的抑损伤指标，且不完全重叠。因此，UTI同间-a胰蛋白酶抑制剂(Ⅰ-a TI)一样，属自然界广泛存在的Kunitz型蛋白酶抑制剂。可抑制糜蛋白酶、a-胰蛋白酶等丝氨酸及多形核中性粒细胞弹性蛋白酶(PMNE)、白细胞组织蛋白酶G、透明质酸、巯基酶等多种酶的作用。
近年来研究显示，UTI有着明显的抗炎性介质作用，UTI可抑制炎症介质释放，预防细胞因子级联反应，抑制白细胞过度激活及炎症介质的释放，阻断炎症介质与白细胞之间的恶性循环作用，减轻各种蛋白酶对缺血-再灌注组织器官的损害，是极强的抗氧化剂。
目的(1)观察围术期双下肢缺血再灌注对血浆炎症因子水平的影响；(2)观察UTI对围术期双下肢缺血再灌注后心肌损伤指标变化的影响；(3)评价UTI给药方式、给药剂量和时机的效果。
方法将30例(男16例，女14例)年龄21～70岁，ASAⅠ级，择期双下肢需上止血带的手术病人，随机分为3组：对照组(C组，n=10)、乌司他丁预处理组(UP组，n=10)、和乌司他丁早期治疗组(UE组，n=10)。UP组：于首次上止血带前15min静脉滴注乌司他丁按6000U／kg，末次松止血带前15min再次静滴6000 U／kg；UE组木次松止血带前15min给予乌司他丁静脉滴注12000U／kg；C组：术次开放止血带前15min静脉滴注等容积生理盐水(均10min内滴完)。三组患者分别于首次上止血带前20min(缺血前)、末次松止血带后30 min(再灌注后30min)、术后(再灌注后)1d、3d和7d于非输液侧肘静脉取血，检测血浆白细胞介素-6(IL-6)、C反应蛋白(CRP)、肿瘤坏死因子-a(TNF-a)、肌酸磷酸激酶(CK-MB)、肌钙蛋白Ⅰ(c-TnI)的水平，并在每个采血的时间点记录呼吸频率(RR)、心率(HR)、平均动脉血压(MBP)等生命体征。
结果
1．一般资料
三组病人的年龄、体重、性别比及上止血带时间、手术时间、术中输液量、输血量、出血量及术后镇痛情况等均无显著性差异(P＞0.05)。
2．生命体征的变化及血管活性药物的应用差异
RR：三组患者各时间点均无显著性差异(P＞0.05)。HR：C组再灌注后30min、1d明显高于缺血前(P＜0.01)；UP组、UE组只在再灌注后30min明显高于缺血前(P＜0.05)，两组与C组比较，再灌注后30min均明显低于后者(P＜0.05)：两组间各时间点未见显著性差异(P＞0.05)。MBP：C组再灌注后30min、1d明显低于缺血前(P＜0.01)；UP组、UE组在再灌注后30min明显低于缺血前(P＜0.01)；再灌注后1d UP组MBP显著高于C组(P＜0.05)，而UE组与C组无明显差异；各时间点UP组、UE组两组差异无统计学意义(P＞0.05)。血管活性药物的使用次数：C组所有患者再灌注后30min、1d给药次数明显高于UP组、UE组(P＜0.05)：UP组、UE组两组间各时间点未见显著性差异(P＞0.05)。
3．血浆炎症介质水平的变化
C组在再灌注后30min、1d、3d血浆IL-6明显高于缺血前(P＜0.01或P＜0.05)，以再灌注后1d最高，UP组、UE组在再灌注后30min、1d、3d血浆IL-6水平也明显升高，但与C组比较，明显低于C组(P＜0.05或P＜0.01)，两组患者再灌注后30min IL-6升至最高，此后逐渐下降；再灌注后UP组IL-6水平虽低于UE组，但差异无统计学意义(P＞0.05)；三组患者再灌注后30min、1d和3d血浆TNF-a水平均明显高于缺血前(P＜0.05或P＜0.01)，但UP组、UE组与C组比较，明显低于后者(P＜0.05或P＜0.01)；再灌注后UP组、UE组各时间点血浆TNF-a在整个围手术期未见显著性差异(P＞0.05)；再灌注后7d三组患者各指标差异无统计学意义(P＞0.05)；C组再灌注后30min、1d、3d、7d CRP均明显高于缺血前(P＜0.01或P＜0.05)，UP组和UE组也明显升高，但明显低于C组(P＜0.01或P＜0.05)，UP组和UE组间比较差异无统计学意义(P＞0.05)，术后7d UE组和UP组CPR恢复正常，C组血浆CPR水平术后7d仍高于缺血前(P＜0.05)。
4．心肌损伤指标的变化
在再灌注后30min、1d、3d，C组血浆CK-MB、c-TnI明显高于缺血前(P＜0.01或P＜0.05)。UP组、UE组血浆CK-MB、c-TnI在再灌注后30min、1d、3d时也有所增高，但与C组相比有显著性差异(P＜0.05或P＜0.01)，C组、UP组、UP组CK-MB、c-TnI均在再灌注后1d升至最高。UP组、UE组各点均无显著性差异(P＞0.05)。再灌注后7d三组患者CK-MB、c-TnI水平均无显著性差异(P＞0.05)。
5．血浆炎症介质与心肌损伤指标的相关分析
IL-6与TNF-a、CRP与CK-MB及c-TnI的相关系数分别为0.880、0.668、0.772、0.670；TNF-a与CRP、CK-MB、c-TnI的相关系数分别为0.712、0.819、0.771；CRP与CK-MB、c-TnI的相关系数分别为0.832、0.799；CK-MB与c-TnI的相关系数为0.825，以上指标两两之间均呈显著正相关(P＜0.01)。
结论
研究结果显示，在缺血再灌注损伤后，三组患者的TNF-a、IL-6、CRP的血浆均有不同程度的升高，但与C组相比，UP组和UE组TNF-a、IL-6、CRP的血浆上升明显受抑，因此可认为UTI可通过抑制炎性介质释放，减少缺血再灌注损伤所致的内环境紊乱。
实验数据显示，三组患者心肌均承受双下肢缺血再关注损伤所致的打击，但与C组相比，UP组和UE组患者血浆CK-MB和c-TnI水平均有显著抑制，证实了在接受UTI治疗后，两组患者的心肌损伤确实在一定程度上获得了缓解。
本实验中血浆CK-MB、c-TnI围术期改变与三组血浆炎性介质的改变情况相似，围术期IL-6、TNF-a、CRP与CK-MB及c-TnI水平变化呈显著的正相关，证实了双下肢IRI后炎症反应在心肌损伤中的作用。
本实验发现，UP组、UE组对炎症因子均有抑制作用，对心肌损伤有明显的保护作用，但对心肌损伤减轻的程度、炎症因子的抑制程度两组无明显差别。
UTI对炎症因子的作用以术后第1d最显著，术后第7d与对照组相比无统计学差异。由于UTI的半衰期较短，仅为40分钟左右，因此对于术后心肌的保护作用还有待于对给药方式的进一步探讨和研究。
本实验发现，UP组、UE组围术期血管活性药物的总使用次数明显低于C组，而三组患者术前的心脏功能无显著差异，提示UTI可能通过减轻炎症反应，改善组织器官的微循环，降低心肌损伤，有利于双下肢缺血再灌低后循环功能的稳定。
实验结果表明，UTI对双下肢缺血再灌注所致远处心肌损伤具有保护作用。其对肢体缺血再灌注所致远处器官心脏损伤的保护作用机理可能主要有两个方面：减轻炎症反应，降低炎症因子对心肌的损伤作用；改善微循环，提高心肌细胞的氧需氧供平衡，保护心肌细胞功能。根据上述研究结果，我们认为UTI值得在围手术期双下肢缺血再灌手术中推广应用。
Limb ischemia-reperfusion(LIR) is a common pathologic process during the perioperative period. High-blooded circulaition is a fundamental warrant for histiocytes to acquire full oxygen and nutrition and to evacuate metabolic products. Any hypoperfusion caused by various factors may induce cell ischemia injury. But the regaining of blood perfusion may cause reperfusion injury (RI). Ischemia-reperfusion injury (IRI) often results in cell metabolism and construction damage more serious. With the development of surgical technique, critical care medicine and preclinical medicine research, the remote organ damage caused by IRI revoke more attention now. LIR may induce remote organ damage, in which the heart is one of the most susceptive organs. Current research points that the IRI not only influence tissue's function and survival, but also involve remote organ, more sevefly it may initiate multiple organ dysfunction and lead to death. LIR may induce heart, lung, kidney and hematological system construction and function damage, in which heart is one of the most important organs involved, including arrhythmia, low blood pressure, lung edema and even circulation collapse. Therefore it is of great clinical value for remote organ to investigate the pathophysiological mechanisms and the clinical intervention of IRI.
Massive study results display that limb IRI is related to increase the oxygen free radical(OFR). After LIRa great deal of OFT, was brought about, and then damaged the limb, meanwhile it activated polymorphonuclear neutrophils(PMNs), release various infammatery intermediates and cytokines to impair directly or indirectly the remote organs. Activated PMNs and its sequent respiratory burst is another cause of remote organs damage induced by IRI. Activated PMNs coaggregated and obstructed the capillary, injured the vasoendothelial cell, produced and released massive OFRs. Experiment results displayed that respiratory burst and OFR release reached to peak after reperfusion 5-30min. Limb ischemia induced by tourniquet mechano-pressurize, surgical trauma, sever tissue damage stirred up OFR production through various pathways. Muscle tissue ischemia and hypoperfusion can generate OFRs through xanthine-xanthine oxidase mechanism, which is the main resource of oxygen free radical. When repel'fusion injury happened, a great deal of cardiac enzymes entered blood circulation, accordingly the changes of cardiac enzyme level could be one of the most sensitive index that indicated myocardium damage caused by LIR. Ischemic skeletal muscle could produce and release a great deal of oxygen free radicals during reperfusion course. The organism removed OFRs via antioxidase system to protect skeletal muscle from ischemia or reperfusion. LIR was the causative factor of inflammatary cascade reaction, and reperfusion might activate PMNs, generate massive oxygen free radicals and inflammatory mediators, and thus the organism was placed in the state of system inflammatary reaction. Surgical ischemia is not only the important cause of local organs injury, but also the cause of remote organ injury such as myocardial injury, which may be mediated by proinflammatary intermediators and cytokines. Higher systemic inflammatary reaction, more severe myocardial damage.
Ulinastatin(UTl) is a broad spectrum protease inhibitor extracted from healthy adult males' urine. As a glycoprotein, it's consisted of 143 amino acids and relative molecular weight is about 67000 dalton. Its primary structure isn't clear yet, but the congener tyrpsin inhibitor primary structure is determined. The basic frame is compromised of two Kunitz regions, and have two activity domains. The two activity domains have broad spectrum for protease inhibition and don't overlap each other. Accordingly, UTI belongs to Kunitz protease inhibitors like I-aTI. It can inhibit serine proteinases such as chymotyrpsin, a-tyrpsin and PMN elastase, leucocyte cathepsin, hyaluronic acid.
Recent studys indicated that UTI had a strong activity to anti-inflammatary mediators. As an effective antioxidant, UTI could inhibit inflammatary mediators release, prevent cytokine cascade reaction, depress leukocyte overact, block up cyclo-action between cytokine and leukocyte and relieve limb IRI aggravated by various proteases.
Objective To observe the effect of both low extremity ischemia reperfusion on the lever of blood plasma inflammation factors, to investigate the protection ofUTI on myocardium damage caused by both low extremity ischemia reperfusion, and to evaluate the effect of UTI with different administration methods, dosage and time.
Method Thirty patients, both low extremities bound with tourniquets during operation, ASA I, were randomly divided into three groups, including operation controlled group (Group C), pretreatment with UTI (Group UP), and early treatment with UTI (Group UE). All the patients were performed with the same anesthesia programe. Patients in Group UP were first administrated with intravenous drop infusion of UTI 6000U/kgat 15min before the first tourniquet bound. The another dosage of UTI 6000U/kg was given 15min before the last tourniquet loosen. UTI was prepared and diluted with 100ml sodium chloride and dropped by intravenous infusion within 10 minutes. Patients in Group UE were administrated with intravenous drop infusion of UTI 12000U/kg at 15min before the last tourniquet loosen. UTI was displaced with the same volume sodium chloride as Group C. The venous blood samples were collected for the measurements of plasma interleukin-6(IL-6), C reactive protein(CRP), tumor necrosis factor-a(TNF-a), creatine phosphokinase(CK-MB) and troponinI(TnI) at 20min before first tourniquet binded(before IRI), 30min after last tourniquet lossen(after IRI 30min) and 1d(after IRI 1d), 3d(after IRI 3d), 7d(after IRI 7d) after the end of operation,. Meanwhile the respiratory rate(RR), heart rate(HR) and mean blood pressure(MBP) were recorded.
Results
General materials
There were no significant difference in three groups at age, weight, sexuality, time of tourniquet, operative time, fluid transfusion volume, blood transfusion and hemorrhage volume(P＞0.05).
Changes of vital signs and use of vasoactive agents
There were no significant differences of RR in each time in three groups(P＞0.05). In Group C, HR was significantly increased at 30min after both lower extremities IRI and 1d after the end of operation, and higher than that before IRI (P＜0.01). In Group UP and UE, HR also rised obviously at 30min after both lower extremities IRI compared with that before IRI (P＜0.05) but lower than that in Group C(P＜0.05). There was no significant difference between Group UP and UE(P＞0.05). Vasoactive agents was more times administrated at 30min after IRI and 1d after the end of operation in group C than that in group UP and UE(P＜0.05). There was no significant difference of the administration fo vasoaxtive agents between Group UP and UE in each time(P＞0.05).
Change of plasma inflammation factors levels
The levels of plasma IL-6, TNF-a and CRP rised significantly in group C at 30min after IRI, 1d and 3d after the end of operation compared with that before IRI, respectively(P＜0.05 or 0.01), and reached to the peak at 1d after the end of operation. The levels of IL-6, TNF-a and CRP in group UP and UE also increased obviously at 30min after IRI, 1d and 3d after the end of operation compared with that before IRI (P＜0.05 or 0.01), but significant lower than in Group C(P＜0.05 or 0.01). There were no obvious difference of TNF-a and CRP between group UP and UE in each time(P＞0.05). All the inflammatary factors nearly returned to the normal level before IRI at 7d after IRI (P＞0.05).
Change of myocardial injury indexes
Plasma CK-MB and c-TnI rised significantly in Group C at 30min after IRI, 1d and 3d after the end of operation compared with that before IRI, respectively(P＜0.05 or 0.01) and reached to the peak at 1d after the end of operation, and also ascended obviously at 30min after IRI, 1d and 3d after the end of operation compared with before IRI (P＜0.05 or 0.01), but significant lower than that in Group C(P＜0.05 or 0.01). There were no obvious difference of plasma CK-MB and c-TnI between Group UP and UE in each time(P＞0.05). Both CK-MB and c-TnI returned to the normal levels at 7d after the end of operation(P＞0.05).
Correlative analysis of inflammation factors and myocardium injury indexes
The correlation coefficients of IL-6 and TNF-a, CRP, CK-MB, c-TnI were 0.880, 0.668, 0.772, 0.670(all P＜0.01), respectively. The correlative coeffcientions of TNF-a and CRP, CK-MB, c-TnI were 0.712, 0.819, 0.771(all P＜0.01), respectively. The correlative coeffcientions of CRP and CK-MB, c-TnI were 0.832 and 0.799(both of P＜0.01), respectively. The correlative coeffcientions of CK-MB and c-TnI was 0.825 (P＜0.01).
Conclusion
The results indicate that both lower extremities ischemia and reperfuion can result in the significantly increased levels of IL-6 and TNF-a in three groups, however, the levels of TNF-a and IL-6 in Group UP and UE was significant lower than that in group C, which hints that UTI can effectively inhibit the releases of proinflammatary factors such as IL-6 and TNF-a, improve the internal deranged environment induced by ischemia-reperfusion, and then effectively protect the remote myocardial injury induced by IRI.
The results display that all the myocardium were injued in a different degree in three groups in the patients with both low extrimeties IRI, but the levels of plasma CK-MB and c-TnI in group UP and UE was significant lower than that in Group C, which infer that the pretreatment or early treatment of UTI can effecively protect the myocardium damage mediated by various proinfammatory factors from both lower extremities ischemia and reperfuison.
The results display that the perioperative changes of CK-MB, c-TnI were similar with those of proinflammatory factors. IL-6, TNF-a, CRP, CK-MB and c-TnI were significantly positively correlated, which confirmed the function of inflammation reaction followed by IRI on myocardium injury.
Our results show that both lower extremities ischemia and reperfusion not only results in the massive releases of proinflammatory mediators and cytokines but also the impairment of remote organ injury such as myocardium. The degree of myocardial injury is correlated with the levels of proinflammatory mediators and cytokines in plasma. The pretreatment and early treatment of UTI can effectively inhibit the releases of proinflammatory mediators and cytokines and protect the impairment of myocardial injury mediated by them from the ischemia and reperfusion. On account of the half life period of UTI is only 40min, so the administration method remains to be further investigated.
In our experimental study, it is also found that that the numbers of times of vasoactive agents was fewer in Group UP and UE than that in Group C. It is suggested that UTI can lighten systemic inflammatory cascade reaction as well as to make for the stablity of systemic circulation and the preotection of myocardium by the improvements of microcirculation of organs. Accordingly, UTI is a valuable drug against the local and remote injury mediated inflammatory reaction and is worth clinical generization and application.
大量研究结果显示，肢体IR损伤与自由基增多有关，肢体缺血再灌注后产生大量氧自由基(OFR)，在损伤肢体的同时，自由基可经其远端效应或直接激活多聚核中性粒细胞(PMN)，使远隔组织器官遭受损伤。PMN活化引发的“呼吸爆发(respiratory burst)”是造成远隔器官损伤的另一原因，激活的PMN相互聚集堵塞微血管，损伤血管内皮细胞，产生并释放大量自由基。实验表明，再灌注后5～30min呼吸爆发氧自由基释放达到高峰。止血带机械性加压致肢体缺血、手术创伤、组织受到严重损害，激发体内多种渠道产生氧自由基，特别是肌肉组织低灌注后缺血组织中通过黄嘌呤-黄嘌呤氧化酶机制，更是产生氧自由基的主要来源。再灌注损伤发生时，出于酶大量进入血液内，故血中酶含量的变化可作为缺血再灌注损伤中肌细胞损害最敏感的一项指标。骨骼肌缺血后的再灌注期产生大量的氧自由基，机体通过抗氧化酶清除氧自由基，而起到保护骨骼肌组织的作用。肢体缺血再灌注是炎症连锁反应的诱发因素，再灌注后可激活中性粒细胞，产生大量氧自由基及炎性介质，使机体处于全身炎症反应状态，引起远隔多器官损伤，除了外科缺血因素以外，炎性因子的水平与心肌损伤之间也是密切相关的，炎症反应越重，心肌损伤也越重，即炎症反应会加重心肌损伤的程度。
乌司他丁(ulinastatin，UTI)是从健康成年男性尿液中提取的广谱蛋白酶抑制剂，是一种糖蛋白，由143个氨基酸组成，相对分子量约为67 000。目前其一级结构尚不清楚，但确定了同类胰蛋白酶抑制剂的一级结构。其基本骨架由两个所谓的Kunitz(库纳斯)型区域组成，具有两个活性功能区，此两个活性区均有很广的抑损伤指标，且不完全重叠。因此，UTI同间-a胰蛋白酶抑制剂(Ⅰ-a TI)一样，属自然界广泛存在的Kunitz型蛋白酶抑制剂。可抑制糜蛋白酶、a-胰蛋白酶等丝氨酸及多形核中性粒细胞弹性蛋白酶(PMNE)、白细胞组织蛋白酶G、透明质酸、巯基酶等多种酶的作用。
近年来研究显示，UTI有着明显的抗炎性介质作用，UTI可抑制炎症介质释放，预防细胞因子级联反应，抑制白细胞过度激活及炎症介质的释放，阻断炎症介质与白细胞之间的恶性循环作用，减轻各种蛋白酶对缺血-再灌注组织器官的损害，是极强的抗氧化剂。
目的(1)观察围术期双下肢缺血再灌注对血浆炎症因子水平的影响；(2)观察UTI对围术期双下肢缺血再灌注后心肌损伤指标变化的影响；(3)评价UTI给药方式、给药剂量和时机的效果。
方法将30例(男16例，女14例)年龄21～70岁，ASAⅠ级，择期双下肢需上止血带的手术病人，随机分为3组：对照组(C组，n=10)、乌司他丁预处理组(UP组，n=10)、和乌司他丁早期治疗组(UE组，n=10)。UP组：于首次上止血带前15min静脉滴注乌司他丁按6000U／kg，末次松止血带前15min再次静滴6000 U／kg；UE组木次松止血带前15min给予乌司他丁静脉滴注12000U／kg；C组：术次开放止血带前15min静脉滴注等容积生理盐水(均10min内滴完)。三组患者分别于首次上止血带前20min(缺血前)、末次松止血带后30 min(再灌注后30min)、术后(再灌注后)1d、3d和7d于非输液侧肘静脉取血，检测血浆白细胞介素-6(IL-6)、C反应蛋白(CRP)、肿瘤坏死因子-a(TNF-a)、肌酸磷酸激酶(CK-MB)、肌钙蛋白Ⅰ(c-TnI)的水平，并在每个采血的时间点记录呼吸频率(RR)、心率(HR)、平均动脉血压(MBP)等生命体征。
结果
1．一般资料
三组病人的年龄、体重、性别比及上止血带时间、手术时间、术中输液量、输血量、出血量及术后镇痛情况等均无显著性差异(P＞0.05)。
2．生命体征的变化及血管活性药物的应用差异
RR：三组患者各时间点均无显著性差异(P＞0.05)。HR：C组再灌注后30min、1d明显高于缺血前(P＜0.01)；UP组、UE组只在再灌注后30min明显高于缺血前(P＜0.05)，两组与C组比较，再灌注后30min均明显低于后者(P＜0.05)：两组间各时间点未见显著性差异(P＞0.05)。MBP：C组再灌注后30min、1d明显低于缺血前(P＜0.01)；UP组、UE组在再灌注后30min明显低于缺血前(P＜0.01)；再灌注后1d UP组MBP显著高于C组(P＜0.05)，而UE组与C组无明显差异；各时间点UP组、UE组两组差异无统计学意义(P＞0.05)。血管活性药物的使用次数：C组所有患者再灌注后30min、1d给药次数明显高于UP组、UE组(P＜0.05)：UP组、UE组两组间各时间点未见显著性差异(P＞0.05)。
3．血浆炎症介质水平的变化
C组在再灌注后30min、1d、3d血浆IL-6明显高于缺血前(P＜0.01或P＜0.05)，以再灌注后1d最高，UP组、UE组在再灌注后30min、1d、3d血浆IL-6水平也明显升高，但与C组比较，明显低于C组(P＜0.05或P＜0.01)，两组患者再灌注后30min IL-6升至最高，此后逐渐下降；再灌注后UP组IL-6水平虽低于UE组，但差异无统计学意义(P＞0.05)；三组患者再灌注后30min、1d和3d血浆TNF-a水平均明显高于缺血前(P＜0.05或P＜0.01)，但UP组、UE组与C组比较，明显低于后者(P＜0.05或P＜0.01)；再灌注后UP组、UE组各时间点血浆TNF-a在整个围手术期未见显著性差异(P＞0.05)；再灌注后7d三组患者各指标差异无统计学意义(P＞0.05)；C组再灌注后30min、1d、3d、7d CRP均明显高于缺血前(P＜0.01或P＜0.05)，UP组和UE组也明显升高，但明显低于C组(P＜0.01或P＜0.05)，UP组和UE组间比较差异无统计学意义(P＞0.05)，术后7d UE组和UP组CPR恢复正常，C组血浆CPR水平术后7d仍高于缺血前(P＜0.05)。
4．心肌损伤指标的变化
在再灌注后30min、1d、3d，C组血浆CK-MB、c-TnI明显高于缺血前(P＜0.01或P＜0.05)。UP组、UE组血浆CK-MB、c-TnI在再灌注后30min、1d、3d时也有所增高，但与C组相比有显著性差异(P＜0.05或P＜0.01)，C组、UP组、UP组CK-MB、c-TnI均在再灌注后1d升至最高。UP组、UE组各点均无显著性差异(P＞0.05)。再灌注后7d三组患者CK-MB、c-TnI水平均无显著性差异(P＞0.05)。
5．血浆炎症介质与心肌损伤指标的相关分析
IL-6与TNF-a、CRP与CK-MB及c-TnI的相关系数分别为0.880、0.668、0.772、0.670；TNF-a与CRP、CK-MB、c-TnI的相关系数分别为0.712、0.819、0.771；CRP与CK-MB、c-TnI的相关系数分别为0.832、0.799；CK-MB与c-TnI的相关系数为0.825，以上指标两两之间均呈显著正相关(P＜0.01)。
结论
研究结果显示，在缺血再灌注损伤后，三组患者的TNF-a、IL-6、CRP的血浆均有不同程度的升高，但与C组相比，UP组和UE组TNF-a、IL-6、CRP的血浆上升明显受抑，因此可认为UTI可通过抑制炎性介质释放，减少缺血再灌注损伤所致的内环境紊乱。
实验数据显示，三组患者心肌均承受双下肢缺血再关注损伤所致的打击，但与C组相比，UP组和UE组患者血浆CK-MB和c-TnI水平均有显著抑制，证实了在接受UTI治疗后，两组患者的心肌损伤确实在一定程度上获得了缓解。
本实验中血浆CK-MB、c-TnI围术期改变与三组血浆炎性介质的改变情况相似，围术期IL-6、TNF-a、CRP与CK-MB及c-TnI水平变化呈显著的正相关，证实了双下肢IRI后炎症反应在心肌损伤中的作用。
本实验发现，UP组、UE组对炎症因子均有抑制作用，对心肌损伤有明显的保护作用，但对心肌损伤减轻的程度、炎症因子的抑制程度两组无明显差别。
UTI对炎症因子的作用以术后第1d最显著，术后第7d与对照组相比无统计学差异。由于UTI的半衰期较短，仅为40分钟左右，因此对于术后心肌的保护作用还有待于对给药方式的进一步探讨和研究。
本实验发现，UP组、UE组围术期血管活性药物的总使用次数明显低于C组，而三组患者术前的心脏功能无显著差异，提示UTI可能通过减轻炎症反应，改善组织器官的微循环，降低心肌损伤，有利于双下肢缺血再灌低后循环功能的稳定。
实验结果表明，UTI对双下肢缺血再灌注所致远处心肌损伤具有保护作用。其对肢体缺血再灌注所致远处器官心脏损伤的保护作用机理可能主要有两个方面：减轻炎症反应，降低炎症因子对心肌的损伤作用；改善微循环，提高心肌细胞的氧需氧供平衡，保护心肌细胞功能。根据上述研究结果，我们认为UTI值得在围手术期双下肢缺血再灌手术中推广应用。
Limb ischemia-reperfusion(LIR) is a common pathologic process during the perioperative period. High-blooded circulaition is a fundamental warrant for histiocytes to acquire full oxygen and nutrition and to evacuate metabolic products. Any hypoperfusion caused by various factors may induce cell ischemia injury. But the regaining of blood perfusion may cause reperfusion injury (RI). Ischemia-reperfusion injury (IRI) often results in cell metabolism and construction damage more serious. With the development of surgical technique, critical care medicine and preclinical medicine research, the remote organ damage caused by IRI revoke more attention now. LIR may induce remote organ damage, in which the heart is one of the most susceptive organs. Current research points that the IRI not only influence tissue's function and survival, but also involve remote organ, more sevefly it may initiate multiple organ dysfunction and lead to death. LIR may induce heart, lung, kidney and hematological system construction and function damage, in which heart is one of the most important organs involved, including arrhythmia, low blood pressure, lung edema and even circulation collapse. Therefore it is of great clinical value for remote organ to investigate the pathophysiological mechanisms and the clinical intervention of IRI.
Massive study results display that limb IRI is related to increase the oxygen free radical(OFR). After LIRa great deal of OFT, was brought about, and then damaged the limb, meanwhile it activated polymorphonuclear neutrophils(PMNs), release various infammatery intermediates and cytokines to impair directly or indirectly the remote organs. Activated PMNs and its sequent respiratory burst is another cause of remote organs damage induced by IRI. Activated PMNs coaggregated and obstructed the capillary, injured the vasoendothelial cell, produced and released massive OFRs. Experiment results displayed that respiratory burst and OFR release reached to peak after reperfusion 5-30min. Limb ischemia induced by tourniquet mechano-pressurize, surgical trauma, sever tissue damage stirred up OFR production through various pathways. Muscle tissue ischemia and hypoperfusion can generate OFRs through xanthine-xanthine oxidase mechanism, which is the main resource of oxygen free radical. When repel'fusion injury happened, a great deal of cardiac enzymes entered blood circulation, accordingly the changes of cardiac enzyme level could be one of the most sensitive index that indicated myocardium damage caused by LIR. Ischemic skeletal muscle could produce and release a great deal of oxygen free radicals during reperfusion course. The organism removed OFRs via antioxidase system to protect skeletal muscle from ischemia or reperfusion. LIR was the causative factor of inflammatary cascade reaction, and reperfusion might activate PMNs, generate massive oxygen free radicals and inflammatory mediators, and thus the organism was placed in the state of system inflammatary reaction. Surgical ischemia is not only the important cause of local organs injury, but also the cause of remote organ injury such as myocardial injury, which may be mediated by proinflammatary intermediators and cytokines. Higher systemic inflammatary reaction, more severe myocardial damage.
Ulinastatin(UTl) is a broad spectrum protease inhibitor extracted from healthy adult males' urine. As a glycoprotein, it's consisted of 143 amino acids and relative molecular weight is about 67000 dalton. Its primary structure isn't clear yet, but the congener tyrpsin inhibitor primary structure is determined. The basic frame is compromised of two Kunitz regions, and have two activity domains. The two activity domains have broad spectrum for protease inhibition and don't overlap each other. Accordingly, UTI belongs to Kunitz protease inhibitors like I-aTI. It can inhibit serine proteinases such as chymotyrpsin, a-tyrpsin and PMN elastase, leucocyte cathepsin, hyaluronic acid.
Recent studys indicated that UTI had a strong activity to anti-inflammatary mediators. As an effective antioxidant, UTI could inhibit inflammatary mediators release, prevent cytokine cascade reaction, depress leukocyte overact, block up cyclo-action between cytokine and leukocyte and relieve limb IRI aggravated by various proteases.
Objective To observe the effect of both low extremity ischemia reperfusion on the lever of blood plasma inflammation factors, to investigate the protection ofUTI on myocardium damage caused by both low extremity ischemia reperfusion, and to evaluate the effect of UTI with different administration methods, dosage and time.
Method Thirty patients, both low extremities bound with tourniquets during operation, ASA I, were randomly divided into three groups, including operation controlled group (Group C), pretreatment with UTI (Group UP), and early treatment with UTI (Group UE). All the patients were performed with the same anesthesia programe. Patients in Group UP were first administrated with intravenous drop infusion of UTI 6000U/kgat 15min before the first tourniquet bound. The another dosage of UTI 6000U/kg was given 15min before the last tourniquet loosen. UTI was prepared and diluted with 100ml sodium chloride and dropped by intravenous infusion within 10 minutes. Patients in Group UE were administrated with intravenous drop infusion of UTI 12000U/kg at 15min before the last tourniquet loosen. UTI was displaced with the same volume sodium chloride as Group C. The venous blood samples were collected for the measurements of plasma interleukin-6(IL-6), C reactive protein(CRP), tumor necrosis factor-a(TNF-a), creatine phosphokinase(CK-MB) and troponinI(TnI) at 20min before first tourniquet binded(before IRI), 30min after last tourniquet lossen(after IRI 30min) and 1d(after IRI 1d), 3d(after IRI 3d), 7d(after IRI 7d) after the end of operation,. Meanwhile the respiratory rate(RR), heart rate(HR) and mean blood pressure(MBP) were recorded.
Results
General materials
There were no significant difference in three groups at age, weight, sexuality, time of tourniquet, operative time, fluid transfusion volume, blood transfusion and hemorrhage volume(P＞0.05).
Changes of vital signs and use of vasoactive agents
There were no significant differences of RR in each time in three groups(P＞0.05). In Group C, HR was significantly increased at 30min after both lower extremities IRI and 1d after the end of operation, and higher than that before IRI (P＜0.01). In Group UP and UE, HR also rised obviously at 30min after both lower extremities IRI compared with that before IRI (P＜0.05) but lower than that in Group C(P＜0.05). There was no significant difference between Group UP and UE(P＞0.05). Vasoactive agents was more times administrated at 30min after IRI and 1d after the end of operation in group C than that in group UP and UE(P＜0.05). There was no significant difference of the administration fo vasoaxtive agents between Group UP and UE in each time(P＞0.05).
Change of plasma inflammation factors levels
The levels of plasma IL-6, TNF-a and CRP rised significantly in group C at 30min after IRI, 1d and 3d after the end of operation compared with that before IRI, respectively(P＜0.05 or 0.01), and reached to the peak at 1d after the end of operation. The levels of IL-6, TNF-a and CRP in group UP and UE also increased obviously at 30min after IRI, 1d and 3d after the end of operation compared with that before IRI (P＜0.05 or 0.01), but significant lower than in Group C(P＜0.05 or 0.01). There were no obvious difference of TNF-a and CRP between group UP and UE in each time(P＞0.05). All the inflammatary factors nearly returned to the normal level before IRI at 7d after IRI (P＞0.05).
Change of myocardial injury indexes
Plasma CK-MB and c-TnI rised significantly in Group C at 30min after IRI, 1d and 3d after the end of operation compared with that before IRI, respectively(P＜0.05 or 0.01) and reached to the peak at 1d after the end of operation, and also ascended obviously at 30min after IRI, 1d and 3d after the end of operation compared with before IRI (P＜0.05 or 0.01), but significant lower than that in Group C(P＜0.05 or 0.01). There were no obvious difference of plasma CK-MB and c-TnI between Group UP and UE in each time(P＞0.05). Both CK-MB and c-TnI returned to the normal levels at 7d after the end of operation(P＞0.05).
Correlative analysis of inflammation factors and myocardium injury indexes
The correlation coefficients of IL-6 and TNF-a, CRP, CK-MB, c-TnI were 0.880, 0.668, 0.772, 0.670(all P＜0.01), respectively. The correlative coeffcientions of TNF-a and CRP, CK-MB, c-TnI were 0.712, 0.819, 0.771(all P＜0.01), respectively. The correlative coeffcientions of CRP and CK-MB, c-TnI were 0.832 and 0.799(both of P＜0.01), respectively. The correlative coeffcientions of CK-MB and c-TnI was 0.825 (P＜0.01).
Conclusion
The results indicate that both lower extremities ischemia and reperfuion can result in the significantly increased levels of IL-6 and TNF-a in three groups, however, the levels of TNF-a and IL-6 in Group UP and UE was significant lower than that in group C, which hints that UTI can effectively inhibit the releases of proinflammatary factors such as IL-6 and TNF-a, improve the internal deranged environment induced by ischemia-reperfusion, and then effectively protect the remote myocardial injury induced by IRI.
The results display that all the myocardium were injued in a different degree in three groups in the patients with both low extrimeties IRI, but the levels of plasma CK-MB and c-TnI in group UP and UE was significant lower than that in Group C, which infer that the pretreatment or early treatment of UTI can effecively protect the myocardium damage mediated by various proinfammatory factors from both lower extremities ischemia and reperfuison.
The results display that the perioperative changes of CK-MB, c-TnI were similar with those of proinflammatory factors. IL-6, TNF-a, CRP, CK-MB and c-TnI were significantly positively correlated, which confirmed the function of inflammation reaction followed by IRI on myocardium injury.
Our results show that both lower extremities ischemia and reperfusion not only results in the massive releases of proinflammatory mediators and cytokines but also the impairment of remote organ injury such as myocardium. The degree of myocardial injury is correlated with the levels of proinflammatory mediators and cytokines in plasma. The pretreatment and early treatment of UTI can effectively inhibit the releases of proinflammatory mediators and cytokines and protect the impairment of myocardial injury mediated by them from the ischemia and reperfusion. On account of the half life period of UTI is only 40min, so the administration method remains to be further investigated.
In our experimental study, it is also found that that the numbers of times of vasoactive agents was fewer in Group UP and UE than that in Group C. It is suggested that UTI can lighten systemic inflammatory cascade reaction as well as to make for the stablity of systemic circulation and the preotection of myocardium by the improvements of microcirculation of organs. Accordingly, UTI is a valuable drug against the local and remote injury mediated inflammatory reaction and is worth clinical generization and application.
引文
1 Hugenholts PC. To reperfuse or not reperfuse which is the question [J]. J Mol Cell Cardiol,1988,20 (2):367-371.
2 陈建常，史振满，王乐农等．肢体缺血再灌注致肺损伤及丹参的保护作用[J]．中国急救医学，2001，21(1)：8-9．
3 丁延峰，周君琳，金国华等．肢体缺血再灌注诱发大鼠心脏血红素氧合酶1的表达[J]．新乡医学院学报，2001，26(18)：77-79．
4 潘成，虞惠康，罗月娥．注射用乌司他丁[J]．中国新药杂志，2000，9(2)：123-125．
5 本间达二．乌司他丁治疗胰腺炎的经验[J]．基础与临床，1983，17(12)：259-263．
6 林晃纪．乌司他丁对失血所致低血压患者的临床作用的研究[J]．急救医学，1998，12(2)：187-192．
7 平山亮夫．乌司他丁治疗胰腺炎的临床效果[J]．新药与临床，1994，33(1)：23-32．
8 Tsujino T, Komastsu Y, Isayama H, et al. Ulinastatin for pancreatitis after endoscopic retrograde cholangiopancreatography: A randomized, controlled trial [J]. Clin Gastroenterol Hepatol,2005, 3(4)376-383.
9 Komori M, Takada K, Tomizawa Y, et al. Urinary trypsin inhibitor improves peripheral microcirculation and bronchospasm associated with systemic anaphalaxis in rabbits in vivo [J]. Shock,2003,20(2): 189-194.
10 Lin SD, Endo R, Sato A, et al. Plasma and urinel evels of urinary trypsin inhibitor in patients with acute and fulminant hepatitis [J]. J Gastroenterol Hepatol, 2002,17(2):140-147.
11 Shimazaki E, TanakaH, OhtaS, et al. Effects of the antiprotease ulinastatin on mortality and oxidant injury in scalded rats[J].Arch Surg, 1995,130(2):994-997.
12 Sato N, Endo S, Kimura Y, et al. Influence of a human protease inhibitor on surgical stress induced immunosupression [J]. Dig Surg,2002,19(4):300-305.
13 Opal SM, DePalo VA. Anti-inflammatory cytokines [J]. Chest,2000,117 (4):1162-1172.
14 Onumata O, Takahashi T, Sato K, et al. Effects of ulinastatin on hypotension and hepatic circμlation in brain dead rabbits [J]. Transplant Proc,2000,32(10):2290-2292.
15 Kashimoto S, Kumazawa T. Effects of urinastatin on cardiac energy metabolism in acute hemorrhage in rats [J].Resuscitation. 1985,14(4):193-197.
16 Nakamura H, Abe S, Shibata Y, et al. Inhibition of neutrophile lastase induced interleukin-8 gene expression by urinary trypsin inhibitor in hum an bronchiale pithelial cells [J]. Int Arch Allergy Immunol,1997,112(7): 157-162.
17 Kawamura T, InadaK, Kimura O, et al. The inhibitory effects of μlinastation on the increase of IL-8 and IL-6 during heart surgery [J]. Masui,1994,43(8):1818-1823.
18 Rothenburger M, Soeparwata R, Deng MC, et al. Prediction of clinical outcome after cardiac surgery: the role of cytokines, endotoxin and antiendotoxin core antibodies [J]. Shock, 2001,16 (Suppl1):44-48.
19 Helmy SA, Wahby MA, El-Nawaway M. The effect of anaesthesia and surgery on plasma cytokine production [J]. Anaesthesia,1999,54(10):733-738.
20 Opal SM , DePalo VA. Anti-inflammatory cytokines [J]. Chest,2000,117 (4):1162-1172.
21 Crozier TA ,Mμller JE, Q uittkat D, et al. Effect of anaesthesia on the cytokine responses to abdominal surgery [J]. Br J Anaesth,1994,72(11):280-286.
22 Natio Y, Tamai S, Sh ingu K, et al. Responses of plasma adrenocort ico tropic hormone, cortisol, and cytokines during and after upper abdominal surgery [J]. Anesthesiology,1999,77(7):426-430.
23 Meldram D. Effect of Ulinastatin on inflammation factors during cardium operation [J]. Am J Physi 1998,274(10):577-579.
24 Sack M. Tumor necrosis factor-alpha in cardiovascu larbiology and the potential role for anti-tumor necrosis factor-alpha therapy in heart disease [J]. Pharmacol Ther, 2002, 94 (1):123-126.
25 Sato Y, Ishikawa S, Otaki A, et al. Induction of acute-phase reactive substances during open-heart surgery and efficacy of ulinastatin [J]. J Thorac Cardiovasc Surg, 2000, 48(7):428-434.
26 Futamura Y, Kajikawa S, Kaga N, et al. Protection against preterm deliver in mice by urinary trypsin inhibitor [J]. Obstet Gynecol,1999, 93(1):100-108.
27 Molor-Erene P, Okajima K, Isobe H, et al. Urinary tyypsin inhibitor reduce LPS-induced hypotension by suppressing tumor necrosis factor-alpha prodfuction through inhibition of HO-1 expression [J]. Am J Physiol Heart Circ Physiol, 2005, 288(3):1265-1271.
28 王振文，尤胜义，王鹏志．大承气冲剂对腹部术后肺功能的影响[J]．中国中西医结合外科杂志，2000，6(4)：237-240．
29 陈贤辉，李之桂，王长鳌，等．C-反应蛋白固相放射免疫分析的改进及初步应用[J]．中国公共卫生学报，1994，13(6)：366-368．
30 王小虎，张卫国，吴清明，等．胃黏膜肥大细胞及C2反应蛋白与Hp致病关系的探讨[J]．中国实验诊断学，2002，6(6)：397-399．
31 杨胜利，何秉贤．C反应蛋白与冠心病[J]．中华心血管病杂志，2001，29(3)：187-189．
32 邵兵，刘琴湘．乌司他丁与心肌缺血再灌注损伤[J]．世界最新医学信息文摘， 2003，2(1)918-922．
33 胡高频， 张文秀， P-选择素与心肌再灌注损伤．国外医学心血管疾病分册，2001，28(1)： 26-29．
34 Mair J. Cardiac troponin Ⅰ and troponin T: are enzymes still relevant as cardiac markers [J]. Clin Chim Acta, 1997,257(1): 115-122.
35 Martins J, LiD J, Baskin LB, et al. Comparison of cardiac troponin Ⅰ and lactate dehydrogenase isoenzymes for the late diagnosis of myocardial injury [J]. Am J Clin Pathol, 1996, 106(6): 705-708.
36 高增栋， 朱健，姚登福．心肌损伤标志物的研究进展[J]．现代医药卫生，2003，19(10)： 1268～1269．
37 于金贵，周广利，孟冬梅，等．乌司他丁对心脏直视手术患者围体外循环期炎性细胞因子和自由基的影响[J]．中华麻醉学杂志，2001，21(6)：21-24．
38 周新，涂植光．临床生物化学和生物化学检验[M]．北京：人民卫生出版社，2003， 282-285．
39 Home BD, Muhlestein JB, Carlquist JF, et al. Statin therapy, lipid levels, C-reactive protein and the survival of patients with angiographically severe coronary artery disease [J]. J Am Coll Cardiol, 2000,36(6):1774-1780.
40 Cain B. The effect of TNF- alpha on apoptosis during cardiopμlmonary bypass [J]. J Surg Res 1998, 76(2):117-123.
41 Tsugiyasuk, Ttakashi T. Interleukin-6 and cardiovascμlar diseases [J]. Jpn Heart J,2003,45(10):183-186
1 刘兴德，陈运贞．蛋白激酶C和蛋白酪氨酸激酶活性对缺血再灌注心肌细胞凋亡影响的实验研究[J]．重庆医科大学学报，2000，20(1)：20-22．
2 刘辉 王成琪 肢体缺血再灌注损伤防治的研究进展 骨与关节损伤杂志 2003 10(18) 713-714．
3 Pararajasingam R, Nichlson ML, Bell PR, et al. Non-cardiogenic pulmonary oedema in vascular surgery [J]. Eur J Vasc Endovasc Surg,1999,17 (2) :933-996
4 曲度，张弦．全腹内脏、下躯、下肢缺血／再灌注损伤动物实验研究[J]杭州医学高等专科学校学报，2002，23(4)：7-10
5 Zhang C, Sheng ZY, Hu S,et al. The role of oxygen-free radical in the apoptosis of enterocytes in scalded rats after delayed resuscitation [J]. J Trauma. 2004, 56 (3) :611-617.
6 Kalia N, Brown NJ, Wood RF, et al. Effects of intestinal ischemia-reperfusion injury on rat peripheral blood neutrophil activation [J]. Dig Dis Sci. 2003, 48 (9):1677-1684.
7 Waisman D, Brod V, Wolff R, et al. Effects of hyperoxia on local and remote microcirculatory inflammatory response after splanchnic ischemia and reperfusion [J]. Am J Physiol Heart Circ Physiol. 2003, 285 (2) : 643-652.
8 Seal JB, Gewertz BL. Vascular dysfunction in ischemia-reperfusion injury [J]. Ann Vasc Surg. 2005,19 (4) :572-584.
9 Dammers R, Wehrens XH, Egbrink MG, et al. Microcirculatory effects of experimental acute limb ischaemia-reperfusion [J]. Br J Surg. 2001, 88 (6) :816-824.
10 Vanniasinkam SH, Crinnon JN, Gough MJ, et al. Post-ischaemic organ dysfunction: a review [J].Eur J Vasc Endovasc Surg,1997,14 (6) :195-198
11 Land WG. The role of postischemic reperfusion injury and other nonantigen-dependent inflammatory pathways in transplantation [J]. Transplantation. 2005,15, 79 (5) :505-514.
12 Waagstein LM, Jivegard, Waljamae H. Hypertionicsal in infusion with thoutdextran70inthere perfusion phaseofex perimentala cute limb ischaemia [J]. Eur J Vasc Endovasc Surg, 1997,13 (5):285-288
13 Blaisdell FW. The pathophysiology of skeletal muscle ischemia and the reperfusion syndrome: a review [J].Cardiovasc Surg. 2002, 10 (6) :620-624
14 刘兴德，袁志柳，黄达枚．双下肢缺血后处理对大鼠心肌缺血再灌注损伤的保护作用[J]．贵阳医学院学报，2004，29(1)：7-9．
15 王强，刘迎龙，朱晓东，等．含抑肽酶低温灌注液减轻体外循环肺损伤[J]．中华胸心血管外科杂志，1999，5(15)：177-179．
1 刘兴德，陈运贞．蛋白激酶C和蛋白酪氨酸激酶活性对缺血再灌注心肌细胞凋亡影响的实验研究[J]．重庆医科大学学报，2000，20(1)：20-22．
2 刘光军，王成琪．肢体缺血再灌注损伤防治的研究进展．骨与关节损伤杂志[J]，2003，18(10)：712-714．
3 Pararajasingam R, Nichlson ML, Bell PR, et al. Non-cardiogenic pulmonary oedema in vascular surgry[J]. Eur J Vasc Endovasc Surg,1999,17(2):933.
4 Adembri C, Kastamoniti E, Bertolozzi Ⅰ, et al. Pulmonary injury follows systemic inflammatory reaction in infrarenal aortic surgery[J|. Crit Care Med. 2004,32(5): 1170-1177.
5 曲度，张弦．常温下腹主动脉阻断动物实验研究-生物多系统时空相关统一论[J]杭州医学高等专科学校学报，2001，4：194-197
6 曲度，王毓．腹内高压综合征，腹腔隙综合征与多脏器危急综合征[J]．江西医学院学报， 2001，4：130-135．
7 Zhang C, Sheng ZY, Hu S,et al. The role of oxygen-free radical in the apoptosis of enterocytes in scalded rats after delayed resuscitation[J]. J Trauma, 2004, 56(3):611-617.
8 Kalia N, Brown NJ, Wood RF, et al. Effects of intestinal ischemia-reperfusion injury on rat peripheral blood neutrophil activation[J]. Dig Dis Sci, 2003 Sep;48(9):1677-1684.
9 Waisman D, Brod V, Wolff R, et al. Effects of hyperoxia on local and remote microcirculatory inflammatory response after splanchnic ischemia and reperfusion[J]. Am J Physiol Heart Circ Physiol, 2003,285(2):643-652.
10 Seal JB, Gewertz BL. Vascular dysfunction in ischemia-reperfusion injury[J]. Ann Vasc Surg, 2005,19(4):572-584.
11 Dammers R, Wehrens XH, oude Egbrink MG, et al. Microcirculatory effects of experimental acute limb ischaemia-reperfusion[J]. Br J Surg, 2001,88(6):816-824.
12 Vanniasinkam SH, Crinnon JN, Gough MJ, et al. Post-ischaemic organ dysfuntion: a review[J]. Eur J Vasc Endovasc Surg,1997,14(2):195.
13 Land WG. The role of postischemic reperfusion injury and other nonantigen-dependent inflammatory pathways in transplantation.Transplantation, 2005,79(5):505-514.
14 Waagstein LM,Jivegard L, Waljamae H. et al Hypertonic saline infusion with or without dextran 70 in the reperfusion phase of experimental acute limb ischaemia.[J].Eur J Vasc Endovasc Surg, 1997,13 (3):285-95.
15 冯峰，冯守减，贾金章．肢体缺血与再灌注损伤的生化代谢及氧自由基改变的实验的研究．中华外科杂志，1990，28(7)：693-696．
16 刘兴德，袁志柳，黄达枚．双下肢缺血后处理对大鼠心肌缺血再灌注损伤的保护作用[J]．贵阳医学院学报，2004，29(1)：7-9
17 Koksel O, Yildirim C, Cinel L, et al. TamerInhibition of poly(ADP-ribose) polymerase attenuates lung tissue damage after hind limb ischemia-reperfusion in rats [J]. Pharmacol Res, 2005,51(5):453-462.
18 Freisleben HJ. Lipoate ameliorates ischemia-reperfusion in animal models [J]. Clin Hemorheol Microcirc, 2000,23(2-4):219-224.
2 陈建常，史振满，王乐农等．肢体缺血再灌注致肺损伤及丹参的保护作用[J]．中国急救医学，2001，21(1)：8-9．
3 丁延峰，周君琳，金国华等．肢体缺血再灌注诱发大鼠心脏血红素氧合酶1的表达[J]．新乡医学院学报，2001，26(18)：77-79．
4 潘成，虞惠康，罗月娥．注射用乌司他丁[J]．中国新药杂志，2000，9(2)：123-125．
5 本间达二．乌司他丁治疗胰腺炎的经验[J]．基础与临床，1983，17(12)：259-263．
6 林晃纪．乌司他丁对失血所致低血压患者的临床作用的研究[J]．急救医学，1998，12(2)：187-192．
7 平山亮夫．乌司他丁治疗胰腺炎的临床效果[J]．新药与临床，1994，33(1)：23-32．
8 Tsujino T, Komastsu Y, Isayama H, et al. Ulinastatin for pancreatitis after endoscopic retrograde cholangiopancreatography: A randomized, controlled trial [J]. Clin Gastroenterol Hepatol,2005, 3(4)376-383.
9 Komori M, Takada K, Tomizawa Y, et al. Urinary trypsin inhibitor improves peripheral microcirculation and bronchospasm associated with systemic anaphalaxis in rabbits in vivo [J]. Shock,2003,20(2): 189-194.
10 Lin SD, Endo R, Sato A, et al. Plasma and urinel evels of urinary trypsin inhibitor in patients with acute and fulminant hepatitis [J]. J Gastroenterol Hepatol, 2002,17(2):140-147.
11 Shimazaki E, TanakaH, OhtaS, et al. Effects of the antiprotease ulinastatin on mortality and oxidant injury in scalded rats[J].Arch Surg, 1995,130(2):994-997.
12 Sato N, Endo S, Kimura Y, et al. Influence of a human protease inhibitor on surgical stress induced immunosupression [J]. Dig Surg,2002,19(4):300-305.
13 Opal SM, DePalo VA. Anti-inflammatory cytokines [J]. Chest,2000,117 (4):1162-1172.
14 Onumata O, Takahashi T, Sato K, et al. Effects of ulinastatin on hypotension and hepatic circμlation in brain dead rabbits [J]. Transplant Proc,2000,32(10):2290-2292.
15 Kashimoto S, Kumazawa T. Effects of urinastatin on cardiac energy metabolism in acute hemorrhage in rats [J].Resuscitation. 1985,14(4):193-197.
16 Nakamura H, Abe S, Shibata Y, et al. Inhibition of neutrophile lastase induced interleukin-8 gene expression by urinary trypsin inhibitor in hum an bronchiale pithelial cells [J]. Int Arch Allergy Immunol,1997,112(7): 157-162.
17 Kawamura T, InadaK, Kimura O, et al. The inhibitory effects of μlinastation on the increase of IL-8 and IL-6 during heart surgery [J]. Masui,1994,43(8):1818-1823.
18 Rothenburger M, Soeparwata R, Deng MC, et al. Prediction of clinical outcome after cardiac surgery: the role of cytokines, endotoxin and antiendotoxin core antibodies [J]. Shock, 2001,16 (Suppl1):44-48.
19 Helmy SA, Wahby MA, El-Nawaway M. The effect of anaesthesia and surgery on plasma cytokine production [J]. Anaesthesia,1999,54(10):733-738.
20 Opal SM , DePalo VA. Anti-inflammatory cytokines [J]. Chest,2000,117 (4):1162-1172.
21 Crozier TA ,Mμller JE, Q uittkat D, et al. Effect of anaesthesia on the cytokine responses to abdominal surgery [J]. Br J Anaesth,1994,72(11):280-286.
22 Natio Y, Tamai S, Sh ingu K, et al. Responses of plasma adrenocort ico tropic hormone, cortisol, and cytokines during and after upper abdominal surgery [J]. Anesthesiology,1999,77(7):426-430.
23 Meldram D. Effect of Ulinastatin on inflammation factors during cardium operation [J]. Am J Physi 1998,274(10):577-579.
24 Sack M. Tumor necrosis factor-alpha in cardiovascu larbiology and the potential role for anti-tumor necrosis factor-alpha therapy in heart disease [J]. Pharmacol Ther, 2002, 94 (1):123-126.
25 Sato Y, Ishikawa S, Otaki A, et al. Induction of acute-phase reactive substances during open-heart surgery and efficacy of ulinastatin [J]. J Thorac Cardiovasc Surg, 2000, 48(7):428-434.
26 Futamura Y, Kajikawa S, Kaga N, et al. Protection against preterm deliver in mice by urinary trypsin inhibitor [J]. Obstet Gynecol,1999, 93(1):100-108.
27 Molor-Erene P, Okajima K, Isobe H, et al. Urinary tyypsin inhibitor reduce LPS-induced hypotension by suppressing tumor necrosis factor-alpha prodfuction through inhibition of HO-1 expression [J]. Am J Physiol Heart Circ Physiol, 2005, 288(3):1265-1271.
28 王振文，尤胜义，王鹏志．大承气冲剂对腹部术后肺功能的影响[J]．中国中西医结合外科杂志，2000，6(4)：237-240．
29 陈贤辉，李之桂，王长鳌，等．C-反应蛋白固相放射免疫分析的改进及初步应用[J]．中国公共卫生学报，1994，13(6)：366-368．
30 王小虎，张卫国，吴清明，等．胃黏膜肥大细胞及C2反应蛋白与Hp致病关系的探讨[J]．中国实验诊断学，2002，6(6)：397-399．
31 杨胜利，何秉贤．C反应蛋白与冠心病[J]．中华心血管病杂志，2001，29(3)：187-189．
32 邵兵，刘琴湘．乌司他丁与心肌缺血再灌注损伤[J]．世界最新医学信息文摘， 2003，2(1)918-922．
33 胡高频， 张文秀， P-选择素与心肌再灌注损伤．国外医学心血管疾病分册，2001，28(1)： 26-29．
34 Mair J. Cardiac troponin Ⅰ and troponin T: are enzymes still relevant as cardiac markers [J]. Clin Chim Acta, 1997,257(1): 115-122.
35 Martins J, LiD J, Baskin LB, et al. Comparison of cardiac troponin Ⅰ and lactate dehydrogenase isoenzymes for the late diagnosis of myocardial injury [J]. Am J Clin Pathol, 1996, 106(6): 705-708.
36 高增栋， 朱健，姚登福．心肌损伤标志物的研究进展[J]．现代医药卫生，2003，19(10)： 1268～1269．
37 于金贵，周广利，孟冬梅，等．乌司他丁对心脏直视手术患者围体外循环期炎性细胞因子和自由基的影响[J]．中华麻醉学杂志，2001，21(6)：21-24．
38 周新，涂植光．临床生物化学和生物化学检验[M]．北京：人民卫生出版社，2003， 282-285．
39 Home BD, Muhlestein JB, Carlquist JF, et al. Statin therapy, lipid levels, C-reactive protein and the survival of patients with angiographically severe coronary artery disease [J]. J Am Coll Cardiol, 2000,36(6):1774-1780.
40 Cain B. The effect of TNF- alpha on apoptosis during cardiopμlmonary bypass [J]. J Surg Res 1998, 76(2):117-123.
41 Tsugiyasuk, Ttakashi T. Interleukin-6 and cardiovascμlar diseases [J]. Jpn Heart J,2003,45(10):183-186
1 刘兴德，陈运贞．蛋白激酶C和蛋白酪氨酸激酶活性对缺血再灌注心肌细胞凋亡影响的实验研究[J]．重庆医科大学学报，2000，20(1)：20-22．
2 刘辉 王成琪 肢体缺血再灌注损伤防治的研究进展 骨与关节损伤杂志 2003 10(18) 713-714．
3 Pararajasingam R, Nichlson ML, Bell PR, et al. Non-cardiogenic pulmonary oedema in vascular surgery [J]. Eur J Vasc Endovasc Surg,1999,17 (2) :933-996
4 曲度，张弦．全腹内脏、下躯、下肢缺血／再灌注损伤动物实验研究[J]杭州医学高等专科学校学报，2002，23(4)：7-10
5 Zhang C, Sheng ZY, Hu S,et al. The role of oxygen-free radical in the apoptosis of enterocytes in scalded rats after delayed resuscitation [J]. J Trauma. 2004, 56 (3) :611-617.
6 Kalia N, Brown NJ, Wood RF, et al. Effects of intestinal ischemia-reperfusion injury on rat peripheral blood neutrophil activation [J]. Dig Dis Sci. 2003, 48 (9):1677-1684.
7 Waisman D, Brod V, Wolff R, et al. Effects of hyperoxia on local and remote microcirculatory inflammatory response after splanchnic ischemia and reperfusion [J]. Am J Physiol Heart Circ Physiol. 2003, 285 (2) : 643-652.
8 Seal JB, Gewertz BL. Vascular dysfunction in ischemia-reperfusion injury [J]. Ann Vasc Surg. 2005,19 (4) :572-584.
9 Dammers R, Wehrens XH, Egbrink MG, et al. Microcirculatory effects of experimental acute limb ischaemia-reperfusion [J]. Br J Surg. 2001, 88 (6) :816-824.
10 Vanniasinkam SH, Crinnon JN, Gough MJ, et al. Post-ischaemic organ dysfunction: a review [J].Eur J Vasc Endovasc Surg,1997,14 (6) :195-198
11 Land WG. The role of postischemic reperfusion injury and other nonantigen-dependent inflammatory pathways in transplantation [J]. Transplantation. 2005,15, 79 (5) :505-514.
12 Waagstein LM, Jivegard, Waljamae H. Hypertionicsal in infusion with thoutdextran70inthere perfusion phaseofex perimentala cute limb ischaemia [J]. Eur J Vasc Endovasc Surg, 1997,13 (5):285-288
13 Blaisdell FW. The pathophysiology of skeletal muscle ischemia and the reperfusion syndrome: a review [J].Cardiovasc Surg. 2002, 10 (6) :620-624
14 刘兴德，袁志柳，黄达枚．双下肢缺血后处理对大鼠心肌缺血再灌注损伤的保护作用[J]．贵阳医学院学报，2004，29(1)：7-9．
15 王强，刘迎龙，朱晓东，等．含抑肽酶低温灌注液减轻体外循环肺损伤[J]．中华胸心血管外科杂志，1999，5(15)：177-179．
1 刘兴德，陈运贞．蛋白激酶C和蛋白酪氨酸激酶活性对缺血再灌注心肌细胞凋亡影响的实验研究[J]．重庆医科大学学报，2000，20(1)：20-22．
2 刘光军，王成琪．肢体缺血再灌注损伤防治的研究进展．骨与关节损伤杂志[J]，2003，18(10)：712-714．
3 Pararajasingam R, Nichlson ML, Bell PR, et al. Non-cardiogenic pulmonary oedema in vascular surgry[J]. Eur J Vasc Endovasc Surg,1999,17(2):933.
4 Adembri C, Kastamoniti E, Bertolozzi Ⅰ, et al. Pulmonary injury follows systemic inflammatory reaction in infrarenal aortic surgery[J|. Crit Care Med. 2004,32(5): 1170-1177.
5 曲度，张弦．常温下腹主动脉阻断动物实验研究-生物多系统时空相关统一论[J]杭州医学高等专科学校学报，2001，4：194-197
6 曲度，王毓．腹内高压综合征，腹腔隙综合征与多脏器危急综合征[J]．江西医学院学报， 2001，4：130-135．
7 Zhang C, Sheng ZY, Hu S,et al. The role of oxygen-free radical in the apoptosis of enterocytes in scalded rats after delayed resuscitation[J]. J Trauma, 2004, 56(3):611-617.
8 Kalia N, Brown NJ, Wood RF, et al. Effects of intestinal ischemia-reperfusion injury on rat peripheral blood neutrophil activation[J]. Dig Dis Sci, 2003 Sep;48(9):1677-1684.
9 Waisman D, Brod V, Wolff R, et al. Effects of hyperoxia on local and remote microcirculatory inflammatory response after splanchnic ischemia and reperfusion[J]. Am J Physiol Heart Circ Physiol, 2003,285(2):643-652.
10 Seal JB, Gewertz BL. Vascular dysfunction in ischemia-reperfusion injury[J]. Ann Vasc Surg, 2005,19(4):572-584.
11 Dammers R, Wehrens XH, oude Egbrink MG, et al. Microcirculatory effects of experimental acute limb ischaemia-reperfusion[J]. Br J Surg, 2001,88(6):816-824.
12 Vanniasinkam SH, Crinnon JN, Gough MJ, et al. Post-ischaemic organ dysfuntion: a review[J]. Eur J Vasc Endovasc Surg,1997,14(2):195.
13 Land WG. The role of postischemic reperfusion injury and other nonantigen-dependent inflammatory pathways in transplantation.Transplantation, 2005,79(5):505-514.
14 Waagstein LM,Jivegard L, Waljamae H. et al Hypertonic saline infusion with or without dextran 70 in the reperfusion phase of experimental acute limb ischaemia.[J].Eur J Vasc Endovasc Surg, 1997,13 (3):285-95.
15 冯峰，冯守减，贾金章．肢体缺血与再灌注损伤的生化代谢及氧自由基改变的实验的研究．中华外科杂志，1990，28(7)：693-696．
16 刘兴德，袁志柳，黄达枚．双下肢缺血后处理对大鼠心肌缺血再灌注损伤的保护作用[J]．贵阳医学院学报，2004，29(1)：7-9
17 Koksel O, Yildirim C, Cinel L, et al. TamerInhibition of poly(ADP-ribose) polymerase attenuates lung tissue damage after hind limb ischemia-reperfusion in rats [J]. Pharmacol Res, 2005,51(5):453-462.
18 Freisleben HJ. Lipoate ameliorates ischemia-reperfusion in animal models [J]. Clin Hemorheol Microcirc, 2000,23(2-4):219-224.
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