输血前血液筛查中新型HBV基因突变及抗原分析的研究
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摘要
研究背景及目的
输血是挽救人类生命的重要手段,输血前对血液进行一系列生物学筛查和安全性评价,对保障安全用血和生命健康具有重要意义。目前我国输血前血液筛查大多仍采用传统的酶联免疫吸附分析(ELISA)检测血液中的病原生物及其成分。近年来随着检测试剂盒灵敏度的不断提高,经输血传播病原微生物的危险性已经处于较低水平。然而,由于病毒感染者“窗口期”献血、隐匿性感染现象与病毒变异的存在,病毒反应性(阳性)献血者的漏检问题依然存在,输血或输注血液制品仍有一定的传播病毒的残余风险,对接受输血者的生命安全造成极大危害。因此探索新的血液筛查方法,采用更加敏感特异方法消除漏检,对于增强输血及血液制品安全性具有重要的实际意义。
乙型肝炎是由感染乙型肝炎病毒(HBV)引起的乙类传染病。我国是乙型肝炎的高发区,一般人群HBV感染率高达57.6%,HBV携带率高达9.09%,HBsAg(乙型肝炎病毒表面抗原)阴性的隐匿性HBV感染在我国普遍存在。HBV感染可引起肝硬化和肝癌,严重危害机体健康和生命安全。因此在输血前血液安全筛查中,乙型肝炎抗原及核酸成分的检测就成为主要靶点。
利用ELISA检测HBsAg是应用最早,开展最广的检测方法,但由于该技术是利用酶催化底物显色的方法,灵敏度较低,操作误差大,易出现假反应性或假非反应性,给血液安全检测带来不确定性。目前我国地市级血站系统采用由双人两次通过ELISA法检测HBV的方法以减少误差,但实际上此方法仅仅是增加了检测的次数,虽可减少操作者方面的实验误差,但不能避免因加样、洗板、变异系数较大等引起的方法学上的固有误差。
近年来,核酸检测技术(NAT, Nucleic Acid Test)在输血前血液筛查中的应用受到高度关注。NAT是直接检测病原体核酸的一系列技术的总称,利用该技术可在病毒感染后数天后即能检出标本中极微量的核酸,大大缩短“窗口期”。NAT敏感性高,与传统的ELISA方法相比,虽然不能完全消除感染“窗口期”但由于病毒核酸转阳之前的血液传染性极低,通过NAT检测,能够明显减少隐匿性感染和病毒变异株,最大限度地预防经输血传播病毒性疾病,因此,NAT可使经输血传播疾病的危险性降到最低,有望成为一种新型的血液筛查传染病的检测技术。
隐匿性HBV感染(OBI)即血清HBsAg阴性而HBV-DNA阳性的HBV感染,表现为病毒载量持续处于低水平复制状态。OBI血液可以通过输血导致受血者感染HBV,其感染率与各地区的HBV感染率和基因型相关。HBsAg阴性的隐匿性HBV感染在我国普遍存在,约有3%的正常人或献血员为HBsAg阴性的HBV携带率者,在单一抗HBc阳性人群中有30%-35%血清HBV DNA阳性,是形成隐匿性乙肝病毒感染的基础。
多种原因可能造成隐匿性HBV感染,位于aa124-147的“a”表位是HBsAg的免疫优势表位,是当前HBsAg检测的主要靶点,HBsAg表位基因突变是导致HBsAg漏检是最主要原因之一。有报道aa100-160氨基酸突变可引起HBsAg抗原活性的明显改变。由于“a”表位也是乙肝疫苗的主要保护性表位以及抗乙肝免疫球蛋白的主要靶点,“a”表位突变可使现有乙肝疫苗及诊断试剂的灵敏度下降,因此研究HBsAg "a"表位对应的HBV S区基因序列具有特别重要的理论和实际意义。
本课题以省级血液中心为研究基地,选择93613份无偿献血标本,采用NAT、基因突变检测和化学发光免疫技术对血液中乙型肝炎病毒的核酸和抗原、抗体联合检测,并与传统的ELISA对比研究；对HBsAg酶联免疫法检测阴性但NAT检测DNA阳性的血清标本进行DNA序列分析和抗体蛋白的确认与分型,探讨HBsAg阴性血清模式与HBV DNA的关系,寻找窗口期和隐匿性HBV感染者；对隐匿性HBV感染者病毒株基因变异进行分析,对病毒株的“a”表位区域进行基因序列测定,分析“a”表位基因突变情况。
本论文旨在了解目前我国HBV筛查体系下献血者隐匿性HBV感染及HBsAg突变株流行状况,建立更为灵敏、准确、符合国际标准要求的输血前血液筛查新模式,并根据筛查结果计算输血传播传染性疾病的残余风险。此外,本文还初步探讨了肝癌细胞PI3K/PKB信号转导通路对细胞凋亡抑制蛋白Survivin变化的影响,旨在为HBV密切相关的肝癌诊断及治疗提供实验依据。
第一部分输血前应用NAT大规模筛查乙肝病毒的模式构建及应用研究
研究方法：
1.核酸检测实验室体系的构建：按照卫生部颁发的《临床基因扩增实验室管理暂行办法》,建立标准核酸检验实验室,之后所有NAT检测均在此实验室进行。
2.不同采血点血液标本的汇集和处理：将不同采血点冷链运送来的标本信息录入采供血及临床输血管理网络体系平台。样品处理：采用血清或抗凝血浆,离心后,取上清用于核酸检测。收集的样品置4℃保存,24小时内进行检测。
3.建立健全样品采集-预处理-保存-使用的标准化流程。标本离心后,经全自动加样仪自动读取标本条码并加样,生成加样文件,发送至实验室服务器的相应加样数据目录下,由全自动酶免分析仪和核酸检测仪处理,将其检测结果相关数据发送至实验室服务器的相应检测数据目录下,实验室信息管理系统(LIS)将对应的加样数据与检测结果相关数据进行处理后,生成每个标本相应检测项目的检测结果释放至血站信息管理系统(Blood Station Information Management System, BIS)。通过深孔板的同步留样,避免人工误差和传统辫血留样错误,配以信息化的管理方式取代人工保存,保证血样检测结果的还原性。
4. ELISA法检测相应的抗原或抗体：ELISA法采用双抗原夹心法或双抗体夹心法检测相应的抗体或抗原。最后在450/630nm处测读吸光度值。按照试剂说明书设立临界值。检测孔A值大于临界值则为反应性,否则为非反应性。酶联免疫法的操作严格按照使用说明书进行,其中核心抗体检测时用生理盐水作1：30稀释。
5.标准NAT检测自动化流程建立：初期采用半自动化设备进行测试,探索出试剂准备、样品核酸提取到检测的适宜条件,最后形成样品自动汇集、检测的标准自动化流程。配套条码扫描,测试过程可全程监控。
6.核酸测定：采用合并检测方式进行。选择ALT阴性,ELISA测定HBsAg阴性、抗HCV Ab阴性、抗HIV Ab阴性的样品以及仅1种ELISA试剂为阴性的标本进行HBV、HCV和HIV病原体核酸的检测。初筛检测方式为合并检测(6个样本混合)。当合并检测汇集池样品中HBV DNA、HCV RNA、HIV RNA任何1项为反应性时,对该汇集池样品再进行拆分检测,挑选出对应的单份反应性样品。合并检测方式同时需满足单份样品临床检测的敏感性要求。
7.NAT体系的方法学评价：采用卫生部临检中心及国际血筛标准品对检测结果的有效性进行评估。当发现检测结果出现偏差时,立即停止试验,查明原因后再继续进行,确保实验结果准确有效。使用该体系对本中心近30000份样本进行连续检测,有效评估实验整体的性能及稳定性。
3.8复检及确认：所有ELISA非反应性,NAT反应性标本都将备份管寄送至中国卫生部临检中心作HBV DNA、HCV RNA和HIV RNA分项定量检测。并对献血者进行随访,以观察是否为抗体检测窗口期标本。
3.9统计学处理
数据处理分析采用SPSS10.0软件进行方差分析,Student't检验来判断差异的统计学意义,P<0.05具有显著性意义。
研究结果
1.NAT检测模式和流程的设计：构建了NAT大规模筛查乙肝病毒的检测模式和流程。
2.NAT检测的数据统计：93613份无偿献血的标本中,经ELISA筛查HBsAg、抗HCV和抗HIV-1/2非反应性及仅1种ELISA试剂为非反应性的标本共91271份,经NAT检测其中有60例反应性标本,反应性检出率为0.066%。
3.NAT反应性标本定量检测：NAT分项鉴别实验反应性率为63.33%(38/60)在我国经血传播疾病病毒酶免试剂阴性核酸试剂阳性献血者标本中,以HBV为主,不同于国外HIV为主的流行模式。
4. HBV DNA反应性标本病毒含量分布：38例HBV DNA反应性标本中病毒含量<20IU/ml的标本28例,占73.68%；20～100IU/ml的标本4例,占10.53%。
5.HBV检测单试剂反应分析：HBV38例单试剂反应性样本中,使用中国产试剂16例,使用美国雅培公司试剂22例；经NAT确认反应性2例,为美国雅培公司试剂单试剂检出。
6.血液筛查核酸检测系统指标：血液筛查核酸检测系统的单项检测灵敏度≥95%,单项检测特异性≥95%,单项检测检测下限是50IU/ml,单项检测下限检出率≥95%。检测通量≥300例样本/每日。
第二部分HBsAg ELISA-/NAT+的HBV血清学模式和病毒变异分析
研究方法
1. HBV DNA提取：使用QIAamp MinElute病毒核酸提取试剂盒提取血清标本中病毒DNA。
2.PCR扩增：PCR扩增使用Prime STAR高保真DNA聚合酶在BIO-RAD IC-Cycler梯度PCR扩增仪或PE2400PCR扩增仪上进行目的片段PCR扩增。引物HBSSWF: AACATCAGGATTCCTAGGAC; HBSSWR:CCTTGTAAGTTGGCGAGAA; HBSS-F TTCATCCTGCTGCTATGCC; HBSS-R:ACGTTTGGTTTTATTAGGGTTC,扩增产物理论上包含HBV pre-S和S区基因。50μl PCR反应体系中加入5×Prime STARTM Buffer (Mg2+plus)10μl, dNTP Mixture(各2.5mM)4μl, Primer1(10μM)1μl, Primer2(10μM)1μl,上述提取的模板DNA2-4μl, Prime STARTM HS DNA Polymerase(2.5U/μl)0.5μl,灭菌蒸馏水加至50μl。PCR反应条件为95℃预变性2min。94℃变性30s,56℃退火30s,72℃延伸55s,共40个循环。72℃2min。反应体系：10×buffer1.0μl, primer F1.0μl, R1.0μl, dNTP1.0μl, Taq0.4μl, ddH2O3.6μl, cDNA2.0μl。每轮PCR均设阴阳性对照,以不同拷贝数的HBV标准质粒为阳性对照,敏感度为50copy/ml。PCR产物取2μl用2%琼脂糖凝胶电泳观察。
3.PCR产物纯化：使用TBE缓冲液制作1.5%琼脂糖凝胶,然后对目的DNA进行琼脂糖凝胶电泳,载样液中加入SYBR Green Ⅰ。对于扩增产物量大(电泳条亮带)的标本扩增50μl体系后电泳,对于扩增产物量小的标本扩增100μl体系后电泳,使用胶纯化试剂盒纯化扩增产物。
4.DNA测序：PCR产物克隆,质粒抽提,使用Big Dye测序反应试剂盒在ABI PRISM3730型全自动DNA测序仪上进行双向测序。
5.HBV基因分型和序列分析：对扩增阳性的PCR产物进行序列测定和进化分析,确定所携HBV的基因型。
6.生物信息学软件处理：测序结果采用ContigExpress、MEGA4生物信息学软件进行分析处理。测得基因片段序列与各基因型参比序列用Blast进行同源列队,比较分析基因突变情况。
研究结果
1.HBV pre-S/S基因PCR扩增结果：56例标本中有41例标本PCR扩增不佳,仅有15例标本PCR产物电泳均可看到1400bp的特异性条带。大部分标本扩增产物量较小,特异性条带较弱。
2.无偿献血者中隐匿性HBV感染情况及基因型分析：23例ELISA HBsAg阳性标本和15例隐匿性HBV标本经巢式PCR扩增阳性检测基因型,结果显示,C基因型在隐匿性HBV中的比例(80.0%)明显高于在HBsAg阳性标本中的比例(17.4%),p<0.05。
3.隐匿性HBV感染者表面抗原S区基因突变分析：3株基因B型和12株基因C型的S区序列分析。有6例在该区域内出现了基因序列点突变,其中有3例为1个碱基点突变,3例发生2个位点的碱基点突变。12例HBV基因C型感染者有4例出现基因点突变,而另3例HBV基因B型感染者中,2例出现基因突变株。
4.HBV S基因“a”决定簇内点突变分析：15例HBsAg ELISA-NAT+标本测序结果表明“a”决定簇内点突变较多,有6例标本存在9个位点处的“a”决定簇内碱基点突变,主要以A-C突变多见。
第三部分ELISA-/NAT+献血者血液HBV抗原抗体确认的研究
研究方法
1. HBsAg ELISA-/NAT+献血者随访策略和随访流程的设计和建立
2.标本采集及处理
3. ECLIA法检测HBV抗体和抗原。
4.NAT检测
5.统计学处理
研究结果
1. HBsAg ELISA-/NAT+献血者随访策略和随访流程的设计和建立。
2. ELISA(-)NAT(+)献血者随访：对60名ELISA(-), NAT(+)献血者立即进行随访,追踪成功26人(34人次)。乙型肝炎病毒表面抗原转阳的有3人；HBV核酸转阴的5人；乙型肝炎病毒表面抗原未转阳但核酸仍为反应性的10人。
3.无偿献血者中HBV窗口期标本追踪检测概况
4.HBsAg ELISA-/NAT+献血者HBV抗体确认。对将59人份核酸检测阳性血液标本进行乙肝“两对半”检测,结果显示,其中有1人HBsAg阳性,HBcAb阳性43人、HBsAb阳性26人,HBcAb单阳性13人。
5.抗-HBc的表达与乙型肝炎隐匿性感染的关联研究：抗-HBc与HBV隐匿性感染有一定的相关性,抗-HBc不仅可以作为既往HBV感染的标志,也可以提示隐匿性HBV感染的可能。
第四部分肝癌细胞survivin表达及bFGF的调控作用
研究方法
1.细胞培养：培养肝癌细胞系(Bel-7402),培养基为含有10%小牛血清的DMEM培养基。每2-3天传代一次。根据文献及预实验结果对将培养的Bel-7402细胞进行不同浓度,不同时间的bFGF或wortmannin处理。
2.细胞处理：将对数生长期细胞1×105/ml,接种于培养瓶,待达到70%融合时换成无血清DMEM培养液孵育过夜,使细胞同步化,然后按时间点和浓度点分组施加因素。
3.Western blot分析：将样品加适量样品悬浮缓冲液后,超声破碎,离心取上清,考马斯亮蓝法测定蛋白浓度。用10%SDS-PAGE电泳后,将PAGE凝胶中的蛋白转移至硝酸纤维素膜。加入一抗(PKB Ser473)进行Western Blot。最后定影、显影,分析结果。
4. RT-PCR检测Bel-7402细胞中survivin的表达。提取细胞总RNA,设计survivin特异性引物,RT-PCR, PCR产物经2%凝胶电泳,凝胶成像仪观察结果并拍照。
5.细胞周期分析：收集消化的贴壁Bel-7402细胞,加入70%冷乙醇固定48h,PI染色,用流式细胞仪进行测定荧光强度。CellQuest分析软件进行细胞周期DNA含量分析。
6.统计学处理
研究结果
1.经bFGF处理后肝癌细胞PKB活性变化
2.经bFGF处理后肝癌细胞survivin mRNA表达的变化
3.PI3K抑制剂对bFGF诱导肝癌细胞survivin mRNA表达的影响。
4. Wortmannin对肝癌细胞凋亡和增殖的影响
研究结论：
1.在输血前乙型肝炎病毒的血液筛查中,采用ELISA技术对血液标本进行初检,NAT技术进行复检的模式,能够对标本进行精确的定性检测,对窗口期的标本和隐匿性肝炎标本具有更强的检出率。
2.目前常规血液筛查中检测HBsAg为阴性的合格血液,仍然存在着窗口期漏检和隐匿性乙肝感染,以隐匿性乙肝为主,病毒株多为C型,且存在着变异株,B型病毒株较少,但突变株较高,突变株可逃避现有的诊断试剂的筛查。
3.抗-HBc与HBV隐匿性感染有一定的相关性,抗-HBc不仅可以作为既往HBV感染的标志,也可以提示隐匿性HBV感染的可能。在不具备开展核酸检测的地区,可以开展抗-HBc检测,以降低输血风险。
4. bFGF通过依赖PI3K/PKB途径调节肝癌细胞Survivin的转录活性,促进其表达,进而抑制肝癌细胞凋亡,PI3K/PKB抑制剂wortmannin可抑制该作用,并导致survivin表达的下调。
创新点
1、以省级血液中心为基础,选择93613例大样本,对现有血液筛查模式下ELISA及NAT对HBV血液筛查的应用价值和输血残余风险进行对比研究。
2、成功构建以核酸检测技术组合酶联免疫筛查技术的输血前经血传播病毒性疾病的检测模式来取代目前单一免疫学检测模式,显著降低经血传播疾病的风险。
3、首次研究了本地区HBV感染者HBV基因类型的分布和变异株的基因序列,丰富了国内HBV基因型分布的数据,为乙肝疫苗研制和乙肝诊断试剂的改进提供实验基础和理论依据,具有重要意义。
4、抗-HBc与HBV隐匿性感染有一定的相关性,抗-HBc不仅可以作为既往HBV感染的标志,也可以提示隐匿性HBV感染的可能,在不具备开展核酸检测的地区,开展抗-HBc检测可降低经血传播疾病的风险。
5.首次证实bFGF刺激Bel-7402细胞后,通过依赖PI3K/PKB途径调节Survivin的转录活性,促进其表达,进而抑制肝癌细胞凋亡。
Background and Objectives
Blood transfusion is an important means to save lives. It is very important to perform a series of biological screening before blood transfusion to make sure blood safety. At present, in our country, the Provincial blood centers and blood stations are using enzyme linked immunosorbent assay (ELISA) for detection of pathogenic microorganisms and their components in blood. With the increasing sensitivity of ELISA kit, spreading risk by blood transfusion is becoming relatively lower. However, due to a viral infection" window period" of blood donation, mutation of the virus and immune-silence phenomenon, virus sero-positive blood donors of missed detection problem is still existed, transfusion of blood or blood products transmitted virus still have some residual risk. So, the usage of blood products transfusion related infection is must to be detected. The safety of blood transfusion is becoming very important points. Therefore, to find the more sensitive and specific method for eliminating positive-undetected, to establish a more perfect mode for blood screening, to enhance the safety of blood products transfusion have important practical significance.
Hepatitis B is kind of serious disease caused by infection with the hepatitis B virus (HBV). At present, in our country, population infection rate is as high as10%-60%. HBsAg negative occult HBV infection is prevalent in our country. HBV infection can cause liver cirrhosis and liver cancer, is seriously harm to the health of the body and life safety. Therefore, the detection of hepatitis B antigen and its nucleic acid components in blood safety screening before transfusion become the main target.
ELISA is using for monitoring HBsAg impost extensive detection method, but its sensitivity is lower, with some operation error, false positive or false negative because of the technique using enzyme catalytic chromogenic substrate method, bring uncertainty to blood safety. At present, in our country, blood center is using double detection by two person through ELISA assay for detection of HBV for reducing the error method, but this method only increased the time of tests, could reduce the operator's experimental error, but could not avoid the methodology error, e.g. repeated sampling and washing plate, and the plate wells differences, coefficient of variation arising from the larger intrinsic error.
In recent years, nucleic acid detection technology (NAT) in blood pretransfusion screening application is highly concerned. The NAT is the directly detection of pathogen nucleic acid. The basic steps include nucleic acid extraction, amplification and detection. NAT is with high sensitivity, can detect trace nucleic acid in sample in a few days after viral infection, which can greatly shorten the" window period". Although NAT theoretically does not completely eliminate infection" window period", but the virus nucleic acid positive before blood infectious is extremely low, can effectively prevent the transfusion transmitted viral diseases. It is said that nucleic acid detection is the last barrier to ensure blood safety. Therefore, the application of NAT can make the transfusion transmitted disease risk to a minimum, so it is expected that NAT will become a new blood screening technology for infectious disease detection.
Occult HBV infection that serum HBsAg-negative HBV infection of HBV-DNA positive performance continued at a low level replication status for viral load. Transfusion recipient can get HBV by infected with OBI, its infection rates relatives with the prevalence of HBV infection and genotypes. HBsAg-negative occult HBV infection prevalent in our country is about3%in the normal. In a single anti-HBc-positive people, the serum DNA positive of HBV is about30%-35%, forming the basis of occult hepatitis B virus infection.
There are a variety of possible causes of occult HBV infection. The "a" epitope at aa124～147is the major immune dominant epitope of HBsAg, and also is the primary target for existing HBsAg detection reagent. HBsAg mutation is the most important for HBsAg reagent undetection. There are many reports on the "a" epitope and the surrounding hydrophilic regions MHR, aa100～160amino acid mutations cause obviously change of HBsAg antigen activity. Since the "a" epitope is also the main target major protective epitope of hepatitis B vaccine, and hepatitis B immune globulin, the popularity of the "a" epitope of mutant may cause the effectiveness of the existing hepatitis B vaccine and diagnostic reagents, HBsAg "a" epitope analysis is particularly important public health significance.
This thesis based on Provincial blood center, selected a large number of samples, using NAT gene mutation detection and chemiluminescence technology joint detection, analyzed Hepatitis B Virus nucleic acid, antigen, antibody in unpaid blood donors, and compared with the traditional ELISA methods. DNA sequence analysis and confirmation of antibody protein typing for negative HBsAg enzyme-linked immunosorbent assay with DNA testing positive serum samples were selected to explore the relationship between serum HBsAg negative mode with HBV DNA, to look for the window period and occult HBV infection. The gene mutation of virus strains was found in occult HBV infection. Than, the virus strains of "a" epitope regions was sequenced, analyzed for the amino acid mutations in the "a" epitope. This thesis is aimed at understanding of HBV occult infection and the prevalence of HBsAg mutant in HBV screening system under the blood donors, to set up a more sensitive, accurate, and consistent with international standards requirements blood screening new model before transfusion, and calculate the residual risk of transfusion-transmitted infectious diseases based on screening results. In addition, the article also discussed the effect of hepatoma cells PI3K/PKB signal transduction pathway to inhibitor of apoptosis protein Survivin, aimed to provide experimental evidence for diagnosis and treatment of liver cancer.
Part One The Construction and application of new mode for HBV nucleic acid detection in large-scale screening before transfusion
Research methods
1.The establishment of NAT lab system:In accordance with the Ministry of Health issued the" Interim Measures for the management of clinical gene amplification laboratory", established an standard nucleic acid testing laboratory. All NAT detection was done in this laboratory.
2. Sample processing:using anticoagulant blood plasma or serum, after centrifugation, supernatant is used for nucleic acid detection. Samples was collected and stored under4℃, tested within24hours.
3. Establish a sample collection-pretreatment-save-the use of standardized processes. Specimen is centrifuged, bar code is read automatically by the automatic pipetting instrument and pipetting to generate a sample file, sent to a laboratory corresponding sample data directory server, automatic ELISA analyzer and nucleic acid detector processing their test results data, corresponding detection data is sent to a lab server directory, LIS processes corresponding sample data and test results data to generate the corresponding test items for each specimen test results and releases to the BIS. Synchronization by deep-well plates to store sample to avoid the risk of errors of human error, and the traditional braided blood storing sample together with the management of information technology to replace the manual save to ensure the reduction of the blood test results.
4. ELISA method to detect the corresponding antigen or antibody:Using a double antigen sandwich method or the double antibody sandwich method to detect the corresponding antigen or antibody. The measured absorbance was read at450/630nm value. Threshold established in accordance with the reagent instructions. The detecting A value greater than the critical value is determined to the reactive or non-reactive. ELISA operation in strict accordance with these instructions, core antibody detection diluted1:30with saline.
5Standard NAT test automation process was established:the initial stage of the experiment using semi-automated equipment testing, a reagent preparation, and sample's nucleic acid extraction to the detection of the entire process. The final formation of automatic sample collection, detection standard is finished for process automation. Support bar code scanning, the testing process can be monitored.
6. NAT:Elected mixing examining mode to detect nucleic acid. All samples were selected from that hepatitis B surface antigen negative, HCV antibody negative, HIV antibody negative samples detected with ELISA. Screening assay for combined detection, sample combination coefficient is6. When combined with the detection collection pool samples HBV DNA, HCV RNA, HIV DNA, any one is positive, the pooled sample pool split detection; pick out the corresponding single positive samples. Combined detection method is carrying on at the same time to meet the single samples in clinical detection sensitivity requirements.
7. The methodological evaluation of NAT system:To assess the effectiveness of the test results under the Ministry of Health rummage center and international blood screening standard. When the deviation of the test results appear, immediately stop the test, to identify the reasons before proceeding, to ensure that the experimental results are accurate and effective. The system is continuously detecting nearly30,000samples for effective assessment of the overall performance and stability of the experiment.
3.8Inspection and confirmation:All ELISA non-reactive, NAT reactive specimens were sent to the Chinese Ministry of Health rummage Center for HBV DNA, HCV RNA and HIV RNA quantitative detection. And blood donors were followed up in order to observe whether the antibody detection window period specimens.
3.9Statistical analysis:SPSS10.0software was used for analysis of variance, Student's test to determine significant differences, P<0.05is significant.
The research results
1. The NAT detection mode and process design:constructed the patterns and processes of NAT large-scale screening for the detection of hepatitis B virus.
2. NAT detection statistics:Among93613donating blood specimens detected by ELISA for HBsAg, anti HCV, anti HIV-1/2, there were total91271for all negative or only one kinds of reagents were negative;91271donating blood specimens were detected by NAT,60cases of positive samples was found, the positive rate was0.066%.
3. Quantitative detection for NAT reactive specimens:NAT breakdown identification test reactivity rate was63.33%(38/60). HBV is the main transmitted disease virus in enzyme immunoassay reagents negative while nucleic acid reagents positive blood donors specimens, different from the HIV as a mainly popular mode in foreign country.
4. The viral content distribution of HBV DNA positive samples:In38cases of HBV DNA positive samples, virus content<20IU/ml specimens is28cases, accounting for73.68%;20～100IU/ml specimens is of4patients, account for10.53%.
5. The result of HBV detection for single reagent detected:38samples in HBV reactive,16cases was tested by reagents made in China,22cases was tested by reagents made in Abbott reagents; NAT to confirm the reactivity of the two cases is for the U.S. Abbott reagents single reagent detection.15cases of HIV single reagent reactivity, reagents nine cases were used by Made in China,6cases were used by French Bio-Rad reagent;1cases detected reactive by NAT was from French Bio-Rad reagent
6. System indicators:Blood screening for nucleic acid detection system of single detection sensitivity was95%, specificity was95%, and single detection limit is50IU/ml, the detection rate of more than95%.
Part Two The analysis of serological mode and mutation of HBsAg in sample of HBV ELISA-/NAT+
Research Methods
1. HBV DNA extraction:extracted the virus DNA from serum samples by the QIAamp MinElute viral nucleic acid extraction kit.
2. PCR amplification:The target fragment was amplified using the Prime STAR high-fidelity DNA polymerase in the Bio-Rad IC-Cycler gradient PCR thermal cycler or PE2400PCR amplification. Primer HBSSWF:AACATCAGGATTCCTAGGAC; HBSSWR:CCTTGTAAGTTGGCGAGAA; HBSS-F: TTCATCCTGCTGCTATGCC; HBSS-R:ACGTTTGGTTTTATTAGGGTTC. Amplification product contains HBV pre-S and S region gene. Add5×Prime STARTM Buffer (Mg2+plus)10μl, dNTP Mixture (2.5mM)4μl, Primer1(10μM)1μl, Primer2(10μM)1μl, the extracted template DNA2～4μl, Prime STARTM HS DNA Polymerase (2.5U/μl)0.5μl, sterile distilled water add to50μl. PCR reaction conditions were95℃denaturation for2min.94℃denaturation for30s,56℃annealing for30s and extension at72℃for55s,40cycles. At72℃for2min. Reaction system:10×buffer1.0μl, primer F1.0μl, primer R1.0μl, dNTP1.0μl, Taq0.4μl, ddH2O3.6μl, cDNA2.0μl. Each round of PCR located negative and positive control, the different HBV standard plasmid copy number as a positive control, a sensitivity of50copies/ml. The PCR product was taken2μl observation using2%agarose gel electrophoresis.
3. PCR product purification:Made1.5%agarose gel by TBE buffer, and then subjected to agarose gel electrophoresis for the analysis of DNA, SYBR Green I was added in the loading solution. If large amount of amplification products obtained, then electrophoresis was done after amplification in50μl system, if small amount of amplification products, then100μl system was used. The amplified product was purified using a gel purification kit for amplification product Lots specimens (electrophoresis bands of light).
4. DNA sequencing:PCR product was cloned, plasmid extraction, bidirectional sequencing using the Big Dye sequencing reaction kit on the ABI PRISM3730type automatic DNA sequencing instrument.
5. HBV genotype and sequence analysis:Sequencing and Phylogenetic Analysis: amplification positive PCR product, identified as carrying the HBV genotype.
6. Bioinformatics software processing:sequencing results using ContigExpress, MEGA4, and bioinformatics software for analysis and processing. The measured gene fragment sequences with the genotype reference sequence homology Blast lined comparative analysis of gene mutation.
The results
1. HBV pre-S/S gene PCR amplification:of56specimens,41specimens PCR amplification poor, only15specimens PCR products can be observed in the1400bp specific bands. The most of specimen's amplification obtained a small amount of product, the specific bands weaker.
2. Occult HBV infection in unpaid donors and genotype analysis:23cases ELISA HBsAg positive samples and15cases of occult HBV specimens was amplified by nested PCR positive test genotype, the results show, C genotype in the occult HBV proportion (80.0%) was significantly higher than the proportion of HBsAg positive samples (17.4%), p<0.05.
3. Occult HBV infection surface antigen S gene mutation analysis:S sequence analysis to three genotype B and12genotype C. Six cases has the gene sequence point mutations in this region, including3cases of a base point mutations occurred,3cases of two base point mutations occurred.12patients with HBV genotype C,4cases point mutation, while the other three cases of HBV genotype B,2cases of gene mutants.
4. HBV S gene "a" decision point mutations in the cluster analysis:15cases of HBsAg ELISA-NAT+specimens sequencing results showed "a" decision cluster has more point mutations, there are nine sites at the "a" determinant within in six specimens, A-C mutation is common.
Part Three HBsAg ELISA-/NAT+donors HBV Ab/Ag recognition and analysis
Research methods
1. The design and building of Follow-up strategies and follow-up process to HBsAg ELISA-/NAT+donors.
2. Specimen collection and handling
3. The ECLIA method to detect HBV antibody and antigen
4. NAT
5. Statistical analysis
The research results
1. The design and building of Follow-up strategies and follow-up process to HBsAg ELISA-/NAT+donors.
2. The follow-up results of ELISA-/NAT+blood donors The HBV antibody confirmation of HBsAg ELISA-/NAT+blood donors:test "two pairs of semi detection" to59cases of nucleic acid testing positive blood samples, the results show, which one HBsAg positive,43HBcAb positive,26people for HBsAb positive,13positive for HBcAb single.
3. HBV window period specimens trace detection profile on unpaid blood donors.
4. The recognition of HBV antibody to HBsAg ELISA-/NAT+blood donors.
5. The association studies between Anti-HBc expression and occult hepatitis B infection:there are certain correlation between anti-HBc and occult HBV infection, anti-HBc not only can be used as the flag of previous HBV infection, but also can prompt the possibility of occult HBV infection.
Part Four The study of survivin expression and regulation in Hepatoma cells Research Methods
1. Cell culture:Hepatoma cell lines (Bel-7402) were cultured, the DMEM medium containing10%fetal calf serum, passed every2-3days. According to the literature and the result of preliminary experiments, Bel-7402cells cultured with different concentrations of bFGF or Wortmannin treatment at different times.
2. Cell treatment:the number of growing cells1×105/ml were seeded in culture flasks, to be up to70%confluence replaced with serum-free DMEM culture medium, incubated overnight, the cell synchronization, and then treat the cell to the point in time and concentration grouping.
3. Western blot analysis:Add appropriate amount of sample suspension buffer to sample, sonication, the centrifugal supernatant, Coomassie-blue method for the determination of protein concentration.10%SDS-PAGE electrophoresis, the PAGE gel, proteins were transferred to nitrocellulose membrane. Join an anti-(PKB Ser473) by Western Blot, developing photo scanning, analysis and processing.
4. RT-PCR method to detect the expression of survivin in Bel-7402cells. Extraction of total RNA, design survivin-specific primers, RT-PCR, PCR products were2%gel electrophoresis gel imager observations and photographed.
4. Cell cycle analysis:Collect adherent cells, digested by adding70%cold ethanol fixation48h. PI staining. The fluorescence intensity was measured by flow cytometry. Use CellQuest analysis software for DNA content analysis of cell cycle.
The results
1. PKB activity increased by bFGF processed in Bel-7402.
2. Survivin mRNA upregulation after bFGF treatment in Bel-7402.
3. PI3K inhibitor wortmannin can inhibit bFGF role of survivin mRNA expression in Bel-7402.
4. Wortmannin promotes apoptosis, inhibition of proliferation in Bel-7402.
Conclusion
1. Hepatitis B virus screening before blood transfusion, the model of the seizure of the blood samples using ELISA technology, NAT technology for re-examination, can precise qualitative and quantitative detection of specimens, and have greater detection rate to window period specimens and occult hepatitis specimens
2. Conventional blood screening for detection of HBsAg negative qualified blood, there is still missed the window period and occult hepatitis B infection. C-type virus strains in occult hepatitis B is in common and mutant B virus trains less but higher mutant. The mutant train is able to evade the existing diagnostic reagent.
3. Anti-HBc related to occult infection, Anti-HBc not only as a sign of previous HBV infection, but also can prompt the testing rate of occult HBV infection in blood screening. Anti-HBc may not only as a sign of previous HBV infection, can be carried out in areas that do no carry out the nucleic acid detection, to reduced risk of blood transfusion.
4. bFGF regulating Survivin transcriptional activity by relying PI3K/PKB ways to promote its expression, thereby inhibiting apoptosis of hepatoma cells, but Wortmannin can inhibit this role and led to the down-regulation of the expression of Survivin.
Points of Innovation
1. Based Provincial Blood Center, selected a large sample, comparatively to study the existing detection technology about enzyme immunoassay and nucleic acid detection in blood screening before transfusion and transfusion residual risk after transfusion.
2. Successfully build a new type of transfusion blood detection mode:compared the enzyme-linked immunosorbent screening technology with nucleic acid detection technology to screen via blood transfusion viral to replace the current single immunological detection methods. It can significantly reduce the risk of transmission through blood.
3. Rich the domestic HBV genotype distribution data understand the northern region family of HBV infection HBV gene type distribution, as well as to determine the progression of the disease and prognosis, suggesting that the antiviral efficacy theory that the northern region of HBV infected with HBV gene type distribution the basis be of great significance.
4. Anti-HBc and HBV occult infection are relative, anti-HBc may not only as a sign of previous HBV infection, but can prompt the possibility of OBI, anti-HBc, in the areas do not have to carry out of nucleic acid detection, can be able to carry out anti-HBc to reduce the risk of disease transmission.
5. The initial recognition bFGF stimulation Bel-7402cells, regulating Survivin transcriptional activity by relying PI3K/PKB ways to promote its expression, thereby inhibiting apoptosis of hepatoma cells.
输血是挽救人类生命的重要手段,输血前对血液进行一系列生物学筛查和安全性评价,对保障安全用血和生命健康具有重要意义。目前我国输血前血液筛查大多仍采用传统的酶联免疫吸附分析(ELISA)检测血液中的病原生物及其成分。近年来随着检测试剂盒灵敏度的不断提高,经输血传播病原微生物的危险性已经处于较低水平。然而,由于病毒感染者“窗口期”献血、隐匿性感染现象与病毒变异的存在,病毒反应性(阳性)献血者的漏检问题依然存在,输血或输注血液制品仍有一定的传播病毒的残余风险,对接受输血者的生命安全造成极大危害。因此探索新的血液筛查方法,采用更加敏感特异方法消除漏检,对于增强输血及血液制品安全性具有重要的实际意义。
乙型肝炎是由感染乙型肝炎病毒(HBV)引起的乙类传染病。我国是乙型肝炎的高发区,一般人群HBV感染率高达57.6%,HBV携带率高达9.09%,HBsAg(乙型肝炎病毒表面抗原)阴性的隐匿性HBV感染在我国普遍存在。HBV感染可引起肝硬化和肝癌,严重危害机体健康和生命安全。因此在输血前血液安全筛查中,乙型肝炎抗原及核酸成分的检测就成为主要靶点。
利用ELISA检测HBsAg是应用最早,开展最广的检测方法,但由于该技术是利用酶催化底物显色的方法,灵敏度较低,操作误差大,易出现假反应性或假非反应性,给血液安全检测带来不确定性。目前我国地市级血站系统采用由双人两次通过ELISA法检测HBV的方法以减少误差,但实际上此方法仅仅是增加了检测的次数,虽可减少操作者方面的实验误差,但不能避免因加样、洗板、变异系数较大等引起的方法学上的固有误差。
近年来,核酸检测技术(NAT, Nucleic Acid Test)在输血前血液筛查中的应用受到高度关注。NAT是直接检测病原体核酸的一系列技术的总称,利用该技术可在病毒感染后数天后即能检出标本中极微量的核酸,大大缩短“窗口期”。NAT敏感性高,与传统的ELISA方法相比,虽然不能完全消除感染“窗口期”但由于病毒核酸转阳之前的血液传染性极低,通过NAT检测,能够明显减少隐匿性感染和病毒变异株,最大限度地预防经输血传播病毒性疾病,因此,NAT可使经输血传播疾病的危险性降到最低,有望成为一种新型的血液筛查传染病的检测技术。
隐匿性HBV感染(OBI)即血清HBsAg阴性而HBV-DNA阳性的HBV感染,表现为病毒载量持续处于低水平复制状态。OBI血液可以通过输血导致受血者感染HBV,其感染率与各地区的HBV感染率和基因型相关。HBsAg阴性的隐匿性HBV感染在我国普遍存在,约有3%的正常人或献血员为HBsAg阴性的HBV携带率者,在单一抗HBc阳性人群中有30%-35%血清HBV DNA阳性,是形成隐匿性乙肝病毒感染的基础。
多种原因可能造成隐匿性HBV感染,位于aa124-147的“a”表位是HBsAg的免疫优势表位,是当前HBsAg检测的主要靶点,HBsAg表位基因突变是导致HBsAg漏检是最主要原因之一。有报道aa100-160氨基酸突变可引起HBsAg抗原活性的明显改变。由于“a”表位也是乙肝疫苗的主要保护性表位以及抗乙肝免疫球蛋白的主要靶点,“a”表位突变可使现有乙肝疫苗及诊断试剂的灵敏度下降,因此研究HBsAg "a"表位对应的HBV S区基因序列具有特别重要的理论和实际意义。
本课题以省级血液中心为研究基地,选择93613份无偿献血标本,采用NAT、基因突变检测和化学发光免疫技术对血液中乙型肝炎病毒的核酸和抗原、抗体联合检测,并与传统的ELISA对比研究；对HBsAg酶联免疫法检测阴性但NAT检测DNA阳性的血清标本进行DNA序列分析和抗体蛋白的确认与分型,探讨HBsAg阴性血清模式与HBV DNA的关系,寻找窗口期和隐匿性HBV感染者；对隐匿性HBV感染者病毒株基因变异进行分析,对病毒株的“a”表位区域进行基因序列测定,分析“a”表位基因突变情况。
本论文旨在了解目前我国HBV筛查体系下献血者隐匿性HBV感染及HBsAg突变株流行状况,建立更为灵敏、准确、符合国际标准要求的输血前血液筛查新模式,并根据筛查结果计算输血传播传染性疾病的残余风险。此外,本文还初步探讨了肝癌细胞PI3K/PKB信号转导通路对细胞凋亡抑制蛋白Survivin变化的影响,旨在为HBV密切相关的肝癌诊断及治疗提供实验依据。
第一部分输血前应用NAT大规模筛查乙肝病毒的模式构建及应用研究
研究方法：
1.核酸检测实验室体系的构建：按照卫生部颁发的《临床基因扩增实验室管理暂行办法》,建立标准核酸检验实验室,之后所有NAT检测均在此实验室进行。
2.不同采血点血液标本的汇集和处理：将不同采血点冷链运送来的标本信息录入采供血及临床输血管理网络体系平台。样品处理：采用血清或抗凝血浆,离心后,取上清用于核酸检测。收集的样品置4℃保存,24小时内进行检测。
3.建立健全样品采集-预处理-保存-使用的标准化流程。标本离心后,经全自动加样仪自动读取标本条码并加样,生成加样文件,发送至实验室服务器的相应加样数据目录下,由全自动酶免分析仪和核酸检测仪处理,将其检测结果相关数据发送至实验室服务器的相应检测数据目录下,实验室信息管理系统(LIS)将对应的加样数据与检测结果相关数据进行处理后,生成每个标本相应检测项目的检测结果释放至血站信息管理系统(Blood Station Information Management System, BIS)。通过深孔板的同步留样,避免人工误差和传统辫血留样错误,配以信息化的管理方式取代人工保存,保证血样检测结果的还原性。
4. ELISA法检测相应的抗原或抗体：ELISA法采用双抗原夹心法或双抗体夹心法检测相应的抗体或抗原。最后在450/630nm处测读吸光度值。按照试剂说明书设立临界值。检测孔A值大于临界值则为反应性,否则为非反应性。酶联免疫法的操作严格按照使用说明书进行,其中核心抗体检测时用生理盐水作1：30稀释。
5.标准NAT检测自动化流程建立：初期采用半自动化设备进行测试,探索出试剂准备、样品核酸提取到检测的适宜条件,最后形成样品自动汇集、检测的标准自动化流程。配套条码扫描,测试过程可全程监控。
6.核酸测定：采用合并检测方式进行。选择ALT阴性,ELISA测定HBsAg阴性、抗HCV Ab阴性、抗HIV Ab阴性的样品以及仅1种ELISA试剂为阴性的标本进行HBV、HCV和HIV病原体核酸的检测。初筛检测方式为合并检测(6个样本混合)。当合并检测汇集池样品中HBV DNA、HCV RNA、HIV RNA任何1项为反应性时,对该汇集池样品再进行拆分检测,挑选出对应的单份反应性样品。合并检测方式同时需满足单份样品临床检测的敏感性要求。
7.NAT体系的方法学评价：采用卫生部临检中心及国际血筛标准品对检测结果的有效性进行评估。当发现检测结果出现偏差时,立即停止试验,查明原因后再继续进行,确保实验结果准确有效。使用该体系对本中心近30000份样本进行连续检测,有效评估实验整体的性能及稳定性。
3.8复检及确认：所有ELISA非反应性,NAT反应性标本都将备份管寄送至中国卫生部临检中心作HBV DNA、HCV RNA和HIV RNA分项定量检测。并对献血者进行随访,以观察是否为抗体检测窗口期标本。
3.9统计学处理
数据处理分析采用SPSS10.0软件进行方差分析,Student't检验来判断差异的统计学意义,P<0.05具有显著性意义。
研究结果
1.NAT检测模式和流程的设计：构建了NAT大规模筛查乙肝病毒的检测模式和流程。
2.NAT检测的数据统计：93613份无偿献血的标本中,经ELISA筛查HBsAg、抗HCV和抗HIV-1/2非反应性及仅1种ELISA试剂为非反应性的标本共91271份,经NAT检测其中有60例反应性标本,反应性检出率为0.066%。
3.NAT反应性标本定量检测：NAT分项鉴别实验反应性率为63.33%(38/60)在我国经血传播疾病病毒酶免试剂阴性核酸试剂阳性献血者标本中,以HBV为主,不同于国外HIV为主的流行模式。
4. HBV DNA反应性标本病毒含量分布：38例HBV DNA反应性标本中病毒含量<20IU/ml的标本28例,占73.68%；20～100IU/ml的标本4例,占10.53%。
5.HBV检测单试剂反应分析：HBV38例单试剂反应性样本中,使用中国产试剂16例,使用美国雅培公司试剂22例；经NAT确认反应性2例,为美国雅培公司试剂单试剂检出。
6.血液筛查核酸检测系统指标：血液筛查核酸检测系统的单项检测灵敏度≥95%,单项检测特异性≥95%,单项检测检测下限是50IU/ml,单项检测下限检出率≥95%。检测通量≥300例样本/每日。
第二部分HBsAg ELISA-/NAT+的HBV血清学模式和病毒变异分析
研究方法
1. HBV DNA提取：使用QIAamp MinElute病毒核酸提取试剂盒提取血清标本中病毒DNA。
2.PCR扩增：PCR扩增使用Prime STAR高保真DNA聚合酶在BIO-RAD IC-Cycler梯度PCR扩增仪或PE2400PCR扩增仪上进行目的片段PCR扩增。引物HBSSWF: AACATCAGGATTCCTAGGAC; HBSSWR:CCTTGTAAGTTGGCGAGAA; HBSS-F TTCATCCTGCTGCTATGCC; HBSS-R:ACGTTTGGTTTTATTAGGGTTC,扩增产物理论上包含HBV pre-S和S区基因。50μl PCR反应体系中加入5×Prime STARTM Buffer (Mg2+plus)10μl, dNTP Mixture(各2.5mM)4μl, Primer1(10μM)1μl, Primer2(10μM)1μl,上述提取的模板DNA2-4μl, Prime STARTM HS DNA Polymerase(2.5U/μl)0.5μl,灭菌蒸馏水加至50μl。PCR反应条件为95℃预变性2min。94℃变性30s,56℃退火30s,72℃延伸55s,共40个循环。72℃2min。反应体系：10×buffer1.0μl, primer F1.0μl, R1.0μl, dNTP1.0μl, Taq0.4μl, ddH2O3.6μl, cDNA2.0μl。每轮PCR均设阴阳性对照,以不同拷贝数的HBV标准质粒为阳性对照,敏感度为50copy/ml。PCR产物取2μl用2%琼脂糖凝胶电泳观察。
3.PCR产物纯化：使用TBE缓冲液制作1.5%琼脂糖凝胶,然后对目的DNA进行琼脂糖凝胶电泳,载样液中加入SYBR Green Ⅰ。对于扩增产物量大(电泳条亮带)的标本扩增50μl体系后电泳,对于扩增产物量小的标本扩增100μl体系后电泳,使用胶纯化试剂盒纯化扩增产物。
4.DNA测序：PCR产物克隆,质粒抽提,使用Big Dye测序反应试剂盒在ABI PRISM3730型全自动DNA测序仪上进行双向测序。
5.HBV基因分型和序列分析：对扩增阳性的PCR产物进行序列测定和进化分析,确定所携HBV的基因型。
6.生物信息学软件处理：测序结果采用ContigExpress、MEGA4生物信息学软件进行分析处理。测得基因片段序列与各基因型参比序列用Blast进行同源列队,比较分析基因突变情况。
研究结果
1.HBV pre-S/S基因PCR扩增结果：56例标本中有41例标本PCR扩增不佳,仅有15例标本PCR产物电泳均可看到1400bp的特异性条带。大部分标本扩增产物量较小,特异性条带较弱。
2.无偿献血者中隐匿性HBV感染情况及基因型分析：23例ELISA HBsAg阳性标本和15例隐匿性HBV标本经巢式PCR扩增阳性检测基因型,结果显示,C基因型在隐匿性HBV中的比例(80.0%)明显高于在HBsAg阳性标本中的比例(17.4%),p<0.05。
3.隐匿性HBV感染者表面抗原S区基因突变分析：3株基因B型和12株基因C型的S区序列分析。有6例在该区域内出现了基因序列点突变,其中有3例为1个碱基点突变,3例发生2个位点的碱基点突变。12例HBV基因C型感染者有4例出现基因点突变,而另3例HBV基因B型感染者中,2例出现基因突变株。
4.HBV S基因“a”决定簇内点突变分析：15例HBsAg ELISA-NAT+标本测序结果表明“a”决定簇内点突变较多,有6例标本存在9个位点处的“a”决定簇内碱基点突变,主要以A-C突变多见。
第三部分ELISA-/NAT+献血者血液HBV抗原抗体确认的研究
研究方法
1. HBsAg ELISA-/NAT+献血者随访策略和随访流程的设计和建立
2.标本采集及处理
3. ECLIA法检测HBV抗体和抗原。
4.NAT检测
5.统计学处理
研究结果
1. HBsAg ELISA-/NAT+献血者随访策略和随访流程的设计和建立。
2. ELISA(-)NAT(+)献血者随访：对60名ELISA(-), NAT(+)献血者立即进行随访,追踪成功26人(34人次)。乙型肝炎病毒表面抗原转阳的有3人；HBV核酸转阴的5人；乙型肝炎病毒表面抗原未转阳但核酸仍为反应性的10人。
3.无偿献血者中HBV窗口期标本追踪检测概况
4.HBsAg ELISA-/NAT+献血者HBV抗体确认。对将59人份核酸检测阳性血液标本进行乙肝“两对半”检测,结果显示,其中有1人HBsAg阳性,HBcAb阳性43人、HBsAb阳性26人,HBcAb单阳性13人。
5.抗-HBc的表达与乙型肝炎隐匿性感染的关联研究：抗-HBc与HBV隐匿性感染有一定的相关性,抗-HBc不仅可以作为既往HBV感染的标志,也可以提示隐匿性HBV感染的可能。
第四部分肝癌细胞survivin表达及bFGF的调控作用
研究方法
1.细胞培养：培养肝癌细胞系(Bel-7402),培养基为含有10%小牛血清的DMEM培养基。每2-3天传代一次。根据文献及预实验结果对将培养的Bel-7402细胞进行不同浓度,不同时间的bFGF或wortmannin处理。
2.细胞处理：将对数生长期细胞1×105/ml,接种于培养瓶,待达到70%融合时换成无血清DMEM培养液孵育过夜,使细胞同步化,然后按时间点和浓度点分组施加因素。
3.Western blot分析：将样品加适量样品悬浮缓冲液后,超声破碎,离心取上清,考马斯亮蓝法测定蛋白浓度。用10%SDS-PAGE电泳后,将PAGE凝胶中的蛋白转移至硝酸纤维素膜。加入一抗(PKB Ser473)进行Western Blot。最后定影、显影,分析结果。
4. RT-PCR检测Bel-7402细胞中survivin的表达。提取细胞总RNA,设计survivin特异性引物,RT-PCR, PCR产物经2%凝胶电泳,凝胶成像仪观察结果并拍照。
5.细胞周期分析：收集消化的贴壁Bel-7402细胞,加入70%冷乙醇固定48h,PI染色,用流式细胞仪进行测定荧光强度。CellQuest分析软件进行细胞周期DNA含量分析。
6.统计学处理
研究结果
1.经bFGF处理后肝癌细胞PKB活性变化
2.经bFGF处理后肝癌细胞survivin mRNA表达的变化
3.PI3K抑制剂对bFGF诱导肝癌细胞survivin mRNA表达的影响。
4. Wortmannin对肝癌细胞凋亡和增殖的影响
研究结论：
1.在输血前乙型肝炎病毒的血液筛查中,采用ELISA技术对血液标本进行初检,NAT技术进行复检的模式,能够对标本进行精确的定性检测,对窗口期的标本和隐匿性肝炎标本具有更强的检出率。
2.目前常规血液筛查中检测HBsAg为阴性的合格血液,仍然存在着窗口期漏检和隐匿性乙肝感染,以隐匿性乙肝为主,病毒株多为C型,且存在着变异株,B型病毒株较少,但突变株较高,突变株可逃避现有的诊断试剂的筛查。
3.抗-HBc与HBV隐匿性感染有一定的相关性,抗-HBc不仅可以作为既往HBV感染的标志,也可以提示隐匿性HBV感染的可能。在不具备开展核酸检测的地区,可以开展抗-HBc检测,以降低输血风险。
4. bFGF通过依赖PI3K/PKB途径调节肝癌细胞Survivin的转录活性,促进其表达,进而抑制肝癌细胞凋亡,PI3K/PKB抑制剂wortmannin可抑制该作用,并导致survivin表达的下调。
创新点
1、以省级血液中心为基础,选择93613例大样本,对现有血液筛查模式下ELISA及NAT对HBV血液筛查的应用价值和输血残余风险进行对比研究。
2、成功构建以核酸检测技术组合酶联免疫筛查技术的输血前经血传播病毒性疾病的检测模式来取代目前单一免疫学检测模式,显著降低经血传播疾病的风险。
3、首次研究了本地区HBV感染者HBV基因类型的分布和变异株的基因序列,丰富了国内HBV基因型分布的数据,为乙肝疫苗研制和乙肝诊断试剂的改进提供实验基础和理论依据,具有重要意义。
4、抗-HBc与HBV隐匿性感染有一定的相关性,抗-HBc不仅可以作为既往HBV感染的标志,也可以提示隐匿性HBV感染的可能,在不具备开展核酸检测的地区,开展抗-HBc检测可降低经血传播疾病的风险。
5.首次证实bFGF刺激Bel-7402细胞后,通过依赖PI3K/PKB途径调节Survivin的转录活性,促进其表达,进而抑制肝癌细胞凋亡。
Background and Objectives
Blood transfusion is an important means to save lives. It is very important to perform a series of biological screening before blood transfusion to make sure blood safety. At present, in our country, the Provincial blood centers and blood stations are using enzyme linked immunosorbent assay (ELISA) for detection of pathogenic microorganisms and their components in blood. With the increasing sensitivity of ELISA kit, spreading risk by blood transfusion is becoming relatively lower. However, due to a viral infection" window period" of blood donation, mutation of the virus and immune-silence phenomenon, virus sero-positive blood donors of missed detection problem is still existed, transfusion of blood or blood products transmitted virus still have some residual risk. So, the usage of blood products transfusion related infection is must to be detected. The safety of blood transfusion is becoming very important points. Therefore, to find the more sensitive and specific method for eliminating positive-undetected, to establish a more perfect mode for blood screening, to enhance the safety of blood products transfusion have important practical significance.
Hepatitis B is kind of serious disease caused by infection with the hepatitis B virus (HBV). At present, in our country, population infection rate is as high as10%-60%. HBsAg negative occult HBV infection is prevalent in our country. HBV infection can cause liver cirrhosis and liver cancer, is seriously harm to the health of the body and life safety. Therefore, the detection of hepatitis B antigen and its nucleic acid components in blood safety screening before transfusion become the main target.
ELISA is using for monitoring HBsAg impost extensive detection method, but its sensitivity is lower, with some operation error, false positive or false negative because of the technique using enzyme catalytic chromogenic substrate method, bring uncertainty to blood safety. At present, in our country, blood center is using double detection by two person through ELISA assay for detection of HBV for reducing the error method, but this method only increased the time of tests, could reduce the operator's experimental error, but could not avoid the methodology error, e.g. repeated sampling and washing plate, and the plate wells differences, coefficient of variation arising from the larger intrinsic error.
In recent years, nucleic acid detection technology (NAT) in blood pretransfusion screening application is highly concerned. The NAT is the directly detection of pathogen nucleic acid. The basic steps include nucleic acid extraction, amplification and detection. NAT is with high sensitivity, can detect trace nucleic acid in sample in a few days after viral infection, which can greatly shorten the" window period". Although NAT theoretically does not completely eliminate infection" window period", but the virus nucleic acid positive before blood infectious is extremely low, can effectively prevent the transfusion transmitted viral diseases. It is said that nucleic acid detection is the last barrier to ensure blood safety. Therefore, the application of NAT can make the transfusion transmitted disease risk to a minimum, so it is expected that NAT will become a new blood screening technology for infectious disease detection.
Occult HBV infection that serum HBsAg-negative HBV infection of HBV-DNA positive performance continued at a low level replication status for viral load. Transfusion recipient can get HBV by infected with OBI, its infection rates relatives with the prevalence of HBV infection and genotypes. HBsAg-negative occult HBV infection prevalent in our country is about3%in the normal. In a single anti-HBc-positive people, the serum DNA positive of HBV is about30%-35%, forming the basis of occult hepatitis B virus infection.
There are a variety of possible causes of occult HBV infection. The "a" epitope at aa124～147is the major immune dominant epitope of HBsAg, and also is the primary target for existing HBsAg detection reagent. HBsAg mutation is the most important for HBsAg reagent undetection. There are many reports on the "a" epitope and the surrounding hydrophilic regions MHR, aa100～160amino acid mutations cause obviously change of HBsAg antigen activity. Since the "a" epitope is also the main target major protective epitope of hepatitis B vaccine, and hepatitis B immune globulin, the popularity of the "a" epitope of mutant may cause the effectiveness of the existing hepatitis B vaccine and diagnostic reagents, HBsAg "a" epitope analysis is particularly important public health significance.
This thesis based on Provincial blood center, selected a large number of samples, using NAT gene mutation detection and chemiluminescence technology joint detection, analyzed Hepatitis B Virus nucleic acid, antigen, antibody in unpaid blood donors, and compared with the traditional ELISA methods. DNA sequence analysis and confirmation of antibody protein typing for negative HBsAg enzyme-linked immunosorbent assay with DNA testing positive serum samples were selected to explore the relationship between serum HBsAg negative mode with HBV DNA, to look for the window period and occult HBV infection. The gene mutation of virus strains was found in occult HBV infection. Than, the virus strains of "a" epitope regions was sequenced, analyzed for the amino acid mutations in the "a" epitope. This thesis is aimed at understanding of HBV occult infection and the prevalence of HBsAg mutant in HBV screening system under the blood donors, to set up a more sensitive, accurate, and consistent with international standards requirements blood screening new model before transfusion, and calculate the residual risk of transfusion-transmitted infectious diseases based on screening results. In addition, the article also discussed the effect of hepatoma cells PI3K/PKB signal transduction pathway to inhibitor of apoptosis protein Survivin, aimed to provide experimental evidence for diagnosis and treatment of liver cancer.
Part One The Construction and application of new mode for HBV nucleic acid detection in large-scale screening before transfusion
Research methods
1.The establishment of NAT lab system:In accordance with the Ministry of Health issued the" Interim Measures for the management of clinical gene amplification laboratory", established an standard nucleic acid testing laboratory. All NAT detection was done in this laboratory.
2. Sample processing:using anticoagulant blood plasma or serum, after centrifugation, supernatant is used for nucleic acid detection. Samples was collected and stored under4℃, tested within24hours.
3. Establish a sample collection-pretreatment-save-the use of standardized processes. Specimen is centrifuged, bar code is read automatically by the automatic pipetting instrument and pipetting to generate a sample file, sent to a laboratory corresponding sample data directory server, automatic ELISA analyzer and nucleic acid detector processing their test results data, corresponding detection data is sent to a lab server directory, LIS processes corresponding sample data and test results data to generate the corresponding test items for each specimen test results and releases to the BIS. Synchronization by deep-well plates to store sample to avoid the risk of errors of human error, and the traditional braided blood storing sample together with the management of information technology to replace the manual save to ensure the reduction of the blood test results.
4. ELISA method to detect the corresponding antigen or antibody:Using a double antigen sandwich method or the double antibody sandwich method to detect the corresponding antigen or antibody. The measured absorbance was read at450/630nm value. Threshold established in accordance with the reagent instructions. The detecting A value greater than the critical value is determined to the reactive or non-reactive. ELISA operation in strict accordance with these instructions, core antibody detection diluted1:30with saline.
5Standard NAT test automation process was established:the initial stage of the experiment using semi-automated equipment testing, a reagent preparation, and sample's nucleic acid extraction to the detection of the entire process. The final formation of automatic sample collection, detection standard is finished for process automation. Support bar code scanning, the testing process can be monitored.
6. NAT:Elected mixing examining mode to detect nucleic acid. All samples were selected from that hepatitis B surface antigen negative, HCV antibody negative, HIV antibody negative samples detected with ELISA. Screening assay for combined detection, sample combination coefficient is6. When combined with the detection collection pool samples HBV DNA, HCV RNA, HIV DNA, any one is positive, the pooled sample pool split detection; pick out the corresponding single positive samples. Combined detection method is carrying on at the same time to meet the single samples in clinical detection sensitivity requirements.
7. The methodological evaluation of NAT system:To assess the effectiveness of the test results under the Ministry of Health rummage center and international blood screening standard. When the deviation of the test results appear, immediately stop the test, to identify the reasons before proceeding, to ensure that the experimental results are accurate and effective. The system is continuously detecting nearly30,000samples for effective assessment of the overall performance and stability of the experiment.
3.8Inspection and confirmation:All ELISA non-reactive, NAT reactive specimens were sent to the Chinese Ministry of Health rummage Center for HBV DNA, HCV RNA and HIV RNA quantitative detection. And blood donors were followed up in order to observe whether the antibody detection window period specimens.
3.9Statistical analysis:SPSS10.0software was used for analysis of variance, Student's test to determine significant differences, P<0.05is significant.
The research results
1. The NAT detection mode and process design:constructed the patterns and processes of NAT large-scale screening for the detection of hepatitis B virus.
2. NAT detection statistics:Among93613donating blood specimens detected by ELISA for HBsAg, anti HCV, anti HIV-1/2, there were total91271for all negative or only one kinds of reagents were negative;91271donating blood specimens were detected by NAT,60cases of positive samples was found, the positive rate was0.066%.
3. Quantitative detection for NAT reactive specimens:NAT breakdown identification test reactivity rate was63.33%(38/60). HBV is the main transmitted disease virus in enzyme immunoassay reagents negative while nucleic acid reagents positive blood donors specimens, different from the HIV as a mainly popular mode in foreign country.
4. The viral content distribution of HBV DNA positive samples:In38cases of HBV DNA positive samples, virus content<20IU/ml specimens is28cases, accounting for73.68%;20～100IU/ml specimens is of4patients, account for10.53%.
5. The result of HBV detection for single reagent detected:38samples in HBV reactive,16cases was tested by reagents made in China,22cases was tested by reagents made in Abbott reagents; NAT to confirm the reactivity of the two cases is for the U.S. Abbott reagents single reagent detection.15cases of HIV single reagent reactivity, reagents nine cases were used by Made in China,6cases were used by French Bio-Rad reagent;1cases detected reactive by NAT was from French Bio-Rad reagent
6. System indicators:Blood screening for nucleic acid detection system of single detection sensitivity was95%, specificity was95%, and single detection limit is50IU/ml, the detection rate of more than95%.
Part Two The analysis of serological mode and mutation of HBsAg in sample of HBV ELISA-/NAT+
Research Methods
1. HBV DNA extraction:extracted the virus DNA from serum samples by the QIAamp MinElute viral nucleic acid extraction kit.
2. PCR amplification:The target fragment was amplified using the Prime STAR high-fidelity DNA polymerase in the Bio-Rad IC-Cycler gradient PCR thermal cycler or PE2400PCR amplification. Primer HBSSWF:AACATCAGGATTCCTAGGAC; HBSSWR:CCTTGTAAGTTGGCGAGAA; HBSS-F: TTCATCCTGCTGCTATGCC; HBSS-R:ACGTTTGGTTTTATTAGGGTTC. Amplification product contains HBV pre-S and S region gene. Add5×Prime STARTM Buffer (Mg2+plus)10μl, dNTP Mixture (2.5mM)4μl, Primer1(10μM)1μl, Primer2(10μM)1μl, the extracted template DNA2～4μl, Prime STARTM HS DNA Polymerase (2.5U/μl)0.5μl, sterile distilled water add to50μl. PCR reaction conditions were95℃denaturation for2min.94℃denaturation for30s,56℃annealing for30s and extension at72℃for55s,40cycles. At72℃for2min. Reaction system:10×buffer1.0μl, primer F1.0μl, primer R1.0μl, dNTP1.0μl, Taq0.4μl, ddH2O3.6μl, cDNA2.0μl. Each round of PCR located negative and positive control, the different HBV standard plasmid copy number as a positive control, a sensitivity of50copies/ml. The PCR product was taken2μl observation using2%agarose gel electrophoresis.
3. PCR product purification:Made1.5%agarose gel by TBE buffer, and then subjected to agarose gel electrophoresis for the analysis of DNA, SYBR Green I was added in the loading solution. If large amount of amplification products obtained, then electrophoresis was done after amplification in50μl system, if small amount of amplification products, then100μl system was used. The amplified product was purified using a gel purification kit for amplification product Lots specimens (electrophoresis bands of light).
4. DNA sequencing:PCR product was cloned, plasmid extraction, bidirectional sequencing using the Big Dye sequencing reaction kit on the ABI PRISM3730type automatic DNA sequencing instrument.
5. HBV genotype and sequence analysis:Sequencing and Phylogenetic Analysis: amplification positive PCR product, identified as carrying the HBV genotype.
6. Bioinformatics software processing:sequencing results using ContigExpress, MEGA4, and bioinformatics software for analysis and processing. The measured gene fragment sequences with the genotype reference sequence homology Blast lined comparative analysis of gene mutation.
The results
1. HBV pre-S/S gene PCR amplification:of56specimens,41specimens PCR amplification poor, only15specimens PCR products can be observed in the1400bp specific bands. The most of specimen's amplification obtained a small amount of product, the specific bands weaker.
2. Occult HBV infection in unpaid donors and genotype analysis:23cases ELISA HBsAg positive samples and15cases of occult HBV specimens was amplified by nested PCR positive test genotype, the results show, C genotype in the occult HBV proportion (80.0%) was significantly higher than the proportion of HBsAg positive samples (17.4%), p<0.05.
3. Occult HBV infection surface antigen S gene mutation analysis:S sequence analysis to three genotype B and12genotype C. Six cases has the gene sequence point mutations in this region, including3cases of a base point mutations occurred,3cases of two base point mutations occurred.12patients with HBV genotype C,4cases point mutation, while the other three cases of HBV genotype B,2cases of gene mutants.
4. HBV S gene "a" decision point mutations in the cluster analysis:15cases of HBsAg ELISA-NAT+specimens sequencing results showed "a" decision cluster has more point mutations, there are nine sites at the "a" determinant within in six specimens, A-C mutation is common.
Part Three HBsAg ELISA-/NAT+donors HBV Ab/Ag recognition and analysis
Research methods
1. The design and building of Follow-up strategies and follow-up process to HBsAg ELISA-/NAT+donors.
2. Specimen collection and handling
3. The ECLIA method to detect HBV antibody and antigen
4. NAT
5. Statistical analysis
The research results
1. The design and building of Follow-up strategies and follow-up process to HBsAg ELISA-/NAT+donors.
2. The follow-up results of ELISA-/NAT+blood donors The HBV antibody confirmation of HBsAg ELISA-/NAT+blood donors:test "two pairs of semi detection" to59cases of nucleic acid testing positive blood samples, the results show, which one HBsAg positive,43HBcAb positive,26people for HBsAb positive,13positive for HBcAb single.
3. HBV window period specimens trace detection profile on unpaid blood donors.
4. The recognition of HBV antibody to HBsAg ELISA-/NAT+blood donors.
5. The association studies between Anti-HBc expression and occult hepatitis B infection:there are certain correlation between anti-HBc and occult HBV infection, anti-HBc not only can be used as the flag of previous HBV infection, but also can prompt the possibility of occult HBV infection.
Part Four The study of survivin expression and regulation in Hepatoma cells Research Methods
1. Cell culture:Hepatoma cell lines (Bel-7402) were cultured, the DMEM medium containing10%fetal calf serum, passed every2-3days. According to the literature and the result of preliminary experiments, Bel-7402cells cultured with different concentrations of bFGF or Wortmannin treatment at different times.
2. Cell treatment:the number of growing cells1×105/ml were seeded in culture flasks, to be up to70%confluence replaced with serum-free DMEM culture medium, incubated overnight, the cell synchronization, and then treat the cell to the point in time and concentration grouping.
3. Western blot analysis:Add appropriate amount of sample suspension buffer to sample, sonication, the centrifugal supernatant, Coomassie-blue method for the determination of protein concentration.10%SDS-PAGE electrophoresis, the PAGE gel, proteins were transferred to nitrocellulose membrane. Join an anti-(PKB Ser473) by Western Blot, developing photo scanning, analysis and processing.
4. RT-PCR method to detect the expression of survivin in Bel-7402cells. Extraction of total RNA, design survivin-specific primers, RT-PCR, PCR products were2%gel electrophoresis gel imager observations and photographed.
4. Cell cycle analysis:Collect adherent cells, digested by adding70%cold ethanol fixation48h. PI staining. The fluorescence intensity was measured by flow cytometry. Use CellQuest analysis software for DNA content analysis of cell cycle.
The results
1. PKB activity increased by bFGF processed in Bel-7402.
2. Survivin mRNA upregulation after bFGF treatment in Bel-7402.
3. PI3K inhibitor wortmannin can inhibit bFGF role of survivin mRNA expression in Bel-7402.
4. Wortmannin promotes apoptosis, inhibition of proliferation in Bel-7402.
Conclusion
1. Hepatitis B virus screening before blood transfusion, the model of the seizure of the blood samples using ELISA technology, NAT technology for re-examination, can precise qualitative and quantitative detection of specimens, and have greater detection rate to window period specimens and occult hepatitis specimens
2. Conventional blood screening for detection of HBsAg negative qualified blood, there is still missed the window period and occult hepatitis B infection. C-type virus strains in occult hepatitis B is in common and mutant B virus trains less but higher mutant. The mutant train is able to evade the existing diagnostic reagent.
3. Anti-HBc related to occult infection, Anti-HBc not only as a sign of previous HBV infection, but also can prompt the testing rate of occult HBV infection in blood screening. Anti-HBc may not only as a sign of previous HBV infection, can be carried out in areas that do no carry out the nucleic acid detection, to reduced risk of blood transfusion.
4. bFGF regulating Survivin transcriptional activity by relying PI3K/PKB ways to promote its expression, thereby inhibiting apoptosis of hepatoma cells, but Wortmannin can inhibit this role and led to the down-regulation of the expression of Survivin.
Points of Innovation
1. Based Provincial Blood Center, selected a large sample, comparatively to study the existing detection technology about enzyme immunoassay and nucleic acid detection in blood screening before transfusion and transfusion residual risk after transfusion.
2. Successfully build a new type of transfusion blood detection mode:compared the enzyme-linked immunosorbent screening technology with nucleic acid detection technology to screen via blood transfusion viral to replace the current single immunological detection methods. It can significantly reduce the risk of transmission through blood.
3. Rich the domestic HBV genotype distribution data understand the northern region family of HBV infection HBV gene type distribution, as well as to determine the progression of the disease and prognosis, suggesting that the antiviral efficacy theory that the northern region of HBV infected with HBV gene type distribution the basis be of great significance.
4. Anti-HBc and HBV occult infection are relative, anti-HBc may not only as a sign of previous HBV infection, but can prompt the possibility of OBI, anti-HBc, in the areas do not have to carry out of nucleic acid detection, can be able to carry out anti-HBc to reduce the risk of disease transmission.
5. The initial recognition bFGF stimulation Bel-7402cells, regulating Survivin transcriptional activity by relying PI3K/PKB ways to promote its expression, thereby inhibiting apoptosis of hepatoma cells.
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