【作者】 高随；

【导师】 宁琴；

【作者基本信息】 华中科技大学 ， 免疫学， 2009， 博士

【摘要】 【背景及目的】HBV感染在全世界范围的流行都非常严重，尤其是在发展中国家和亚太地区，我国是HBV感染高发地区，人群携带率高达约10～20％。在亚洲，重型肝炎最主要是由HBV感染引起的，而在欧美国家重型肝炎主要是由酒精性肝炎和药物性肝炎引起。重型肝炎是急骤发生广泛性肝细胞坏死而引起重度肝功能障碍，出现以肝性脑病为主的肝功能衰竭症状之高危疾病。该病病情严重，发展迅猛，发病机制尚不明了，临床缺乏上特异、有效的治疗靶点和干预手段，除非实施紧急肝移植，绝大部分患者预后不良。因此探索重型肝炎肝细胞死亡机制，并开发针对其发病机制、阻断病理衍变过程的基因治疗手段正在成为该领域的研究热点和发展方向。肝细胞过度凋亡引起的肝损伤可以引起肝衰竭，并在多种肝病的发生过程中起重要作用。在生理状态下发生的凋亡主要是清除那些损伤的和多余的细胞，凋亡小体的吞噬不伴随炎症反应的发生。而在病理状态下，肝细胞凋亡则有可能引起炎症反应，出现中性粒细胞的浸润、肝星状细胞活化，发生肝脏纤维化。在重型肝炎的发病过程中，Fas与TNFα系统的激活会促进肝细胞凋亡的发生。肝细胞的凋亡途径包括胞膜上死亡受体介导的外部途径和线粒体介导的内部途径，肝细胞膜上表达最广泛的死亡受体是CD95(AP021PFas)和TNF2a受体1(CD120a)。多项研究表明，急慢性肝病中肝细胞的凋亡一般由死亡受体介导。肝细胞对CD120a和CD95介导的凋亡具有高度的敏感性。体外实验也证实了这一点。在LPS+D-GalN诱导的爆发性肝衰竭模型中，LPS的毒性实际上是由于严重的凋亡性肝损伤和肝细胞完全破坏所引起的。然而，有关肝细胞凋亡在MHV-3诱导的小鼠重型肝炎肝衰竭中的重要作用还未见相关文献报道，因此研究肝细胞凋亡及其调控机制对重型肝炎肝损伤发生机理的理解具有重要意义。TNFα是一种由巨噬细胞产生的细胞因子。TNFα对不同的细胞类型发挥各种不相同的作用，在许多重要的病理和生理过程下作为一重要的介质存在。而且TNFα还是凋亡的重要介质之一，TNFα的大多数生物学效应TNFR1介导。TNFR1胞内区含死亡结构域，可引起细胞凋亡或者通过激活核转录因子NF-KB使某些细胞增殖、分化，另外也可触发信号传导级联而导致细胞凋亡。RNA干扰(RNA interference，RNAi)是指特异性针对目标基因的同源双链RNA(double-strain RNA，dsRNA)的导入引起目的基因不表达或减效表达，具有高效、特异，快速等优点，应用前景广阔。前期研究发现通过尾静脉高压注射可以使质粒[DNA在小鼠肝脏高效表达。尾静脉高压注射可将大体积的裸质粒DNA溶液经小鼠尾静脉快速注入，大量的质粒溶液导致循环血量的急剧增加，超过心脏负荷，血液积聚在肝窦中不能回流，延长了质粒DNA在肝窦中的停留时间，从而被肝组织细胞摄取。因此该方法可以广泛应用于肝脏疾病基因干预的研究。从基因水平沉默“有害”高表达的基因可以更好地阐明该基因在疾病的发生发展中重要作用，也为探索人类重型肝炎临床治疗方法提供新的思路和手段。具体研究目的如下：1．针对重型肝炎发病关键基因mTNFR1构建siRNA干扰质粒，在中国仓鼠卵巢细胞(CH0)中可以明显抑制TNFR1基因的表达。2．研究mTNFR1shRNA干扰质粒对重型肝炎小鼠体内mTNFR1表达的抑制，改善肝细胞的凋亡情况，生化指标以及对小鼠重型肝炎病情发展的影响。3．针对凋亡相关基因Fas，TNFR1构建其真核表达载体和microRNA表达载体，并研究在miRNA在细胞水平对基因表达的抑制作用。【方法】1．构建mTNFR1shRNA干扰质粒和非相关对照质粒，细胞水平分别共转染mTNFR1shRNA干扰质粒和pEGFP-TNFR1，mTNFR1shRNA干扰质粒和pCDNA3．0-TNFR1，并通过显微镜下观察荧光，RT-PCR、western blot技术检测mTNFR1shRNA干扰质粒的体外干预效应。2．采用MHV-3感染Balb／cJ小鼠制造重型肝炎动物模型；通过尾静脉高压注射将目的基因导入小鼠肝脏，并检测目的基因在肝脏的表达效率；mTNFR1shRNA干扰质粒高压注射后，检测小鼠肝组织病理、血清生化学变化，并观察重型肝炎小鼠生存率的改变；通过Real-time PCR、免疫组化检测mTNFR1shRNA干扰质粒干预后的小鼠肝脏mTNFR1的表达情况；用TUNNEL法检测肝细胞凋亡情况的改变，并计算凋亡指数。3．构建人类Fas和TNFR1基因的真核表达载体及其miRNA表达载体，并将其共转染至人293T细胞，通过Real-time PCR和western blot检测在细胞水平对基因表达的抑制作用。【结果】1．成功构建mTNFR1shRNA干扰质粒和非相关对照质粒，并鉴定无误；分别共转染mTNFR1shRNA干扰质粒和pEGFP-TNFR1，mTNFR1shRNA干扰质粒和pCDNA3．0-TNFR1至CHO细胞，镜下可见干预后荧光明显减弱，RT-PCR、Western blot结果证实mTNFR1shRNA干扰质粒细胞水平显著抑制mTNFRl的表达。2．尾静脉高压注射可以将目的基因导入小鼠肝脏，24h重复注射可以使表达效率提高到40％。mTNFR1shRNA干扰质粒高压注射后，重型肝炎小鼠生存率从0提高到13．3％，并显著改善肝组织病理学变化和血清学指标；mTNFR1shRNA干扰质粒高压注射后，显著抑制mTNFR1在重型肝炎小鼠模型体内的表达，并显著减少肝细胞的凋亡。3．成功构建人类凋亡相关基因Fas，TNFR1的真核表达载体和miRNA干扰质粒，并鉴定无误；Fas-miRNA和TNFR1-miRNA干扰质粒在细胞水平显著抑制hFas和hTNFR1的表达。【结论】1．本研究利用在线软件设计合成针对小鼠TNFR1基因的RNA干扰靶序列，通过PCR将其连接于pMSCV-U6载体，并通过序列鉴定无误。构建成功的的mTNFR1 shRNA干扰质粒通过转染CH0细胞，体外实验证实可以有效且特异地下调目的基因的表达。2．建立MHV-3诱导的重型肝炎小鼠模型，并通过尾静脉高压注射技术将外源基因高效导入小鼠肝脏，mTNFR1 shRNA干扰质粒通过下调小鼠肝脏TNFR1基因的表达可以显著提高小鼠的生存时间和生存率，并且生化指标和肝脏的炎症均得到明显改善。为今后重型肝炎、肿瘤、代谢性疾病等凋亡相关的疾病治疗提供了一条新途径。3．重型肝炎的肝细胞死亡机制主要包括肝细胞的过度凋亡，因此基因治疗的靶点需包含与肝细胞凋亡相关的多个关键基因。本研究构建了凋亡相关基因Fas和TNFR1真核表达质粒和microRNA干扰质粒，通过转染至人293T细胞，在体外试验中有效且特异得下调目的基因的表达。更多还原

【Abstract】 【BACKGROUND&OBJECTIVE】Worldwide about 400 million people are chronic infection of hepatitis B virus (HBV),especially in Asia,where there is an HBV chronic carrier rate of 10-20%.In the Far East,fulminant hepatic failure is mainly due to viral hepatitis.The mortality of fulminant viralhepatitis is over 80% in the cases of lack of immediate liver transplantation.The fulminanthepatitis is characterized by recurrent flares of hepatocellular injury,resulting inwidespread or total hepatocellular necrosis and apoptosis in weeks or even days,andoccurrence of hepatic encephalopathy.The mechanisms for virus-induced hepatocyte injuryare still not clearly known.Due to the lack of specific and effective clinical treatment,unless an emergency liver transplant,the majority of patients are with poor prognosis.Therefore investigating the mechanism of hepatocytes death in severe hepatitis,anddeveloping gene therapeutic means with which we can block the pathological process ofthis servere disease has become a serious issue in this field.Hepatocytes apoptosis occurred in the liver plays an important role in the process ofmany liver diseases,especially hepatic failure caused by various reasons.In the process ofsevere hepatitis,Fas and TNF system activated will affect the severity of hepatocytesapoptosis.Actually LPS toxicity is due to serious apoptosis that further induce liver injury and destruction in the FHF model induced by LPS/D-GalN.However the contribution ofhepatocytes apoptosis to the development of murine hepatitis virus strain 3 (MHV-3)induced hepatic failure in Balb/cJ mice has never been explored,which in turn might beone of the important aspects for understanding the pathogenesis of hepatocytes injury.TNFαis one of the cytokines which excreted by macrophage.TNFα/TNFR inducedapoptotic death of hepatocytes may also be of major importance under conditions causingacute liver failure in septic shock.In fact,hepatic failure and tissue destruction as a result ofendogenously produced TNF has recently been demonstrated to be mediated by the 55-kDaTNF receptor (TNFR1).There has been significant interest in the use of RNA interference (RNAi) inhibition ofgene expression.RNAi is thought to catalytically trigger degradation of target mRNAsbefore translation.It is therefore possible that RNAi could act synergistically to specificallyresult in more efficient gene silencing.Target gene silence can be applied to evaluate therole and importance of the target gene in the pathogenesis of interested disease.Currentlyhigh levels of gene transfer to mouse liver could be achieved by tail vein hydrodynamicinjection of plasmid DNA solution in a large volume.It has been proposed that the injectedDNA solution accumulates mainly in the liver because of its flexible structure and bloodflow cease induced by transient heart failure,which can accommodate large volume ofsolution,and the hydrostatic pressure forces plasimid DNA into the liver cells before it ismixed with blood.This technology has been extensively used in gene therapy for mousestudies.Therefore the purposes of this study are as the follows:1.To construct a mTNFR1 shRNA plasmid which can inhibit the expression of mTNFR1in CHO cells,a Chinese hamster ovary cell line.2.To investigate the effect of mTNFR1 shRNA plasmid on mTNFR1 expression in vivoand the disease progress in MHV-3 induced fulminant hepatitis mice model.3.To construct the eukaryotic expression vectors of human fgl2,Fas and TNFR1 geneand miRNA expression plasmids of hFas and hTNFR1 named p-hFasmiRNA andp-hTNFRⅠmiRNA,which can inhibit the expression of hFas and hTNFRⅠin 293Tcell lines. 【METHODS】1.The mTNFR1shRNA plasmid and irrelative shRNA plasmid as a control wereconstructed.CHO cells were transfected with mTNFR1shRNA plasmid,interventioneffect of mTNFR1shRNA plasmid in vitro was detected by RT-PCR and Western-blot.Target gene was introduced into mice liver by hydrodynamic injections,and expressionefficiency of target gene was detected;After hydrodynamic injection ofmTNFR1shRNA plasmid,the survival rate of mice,hepatic pathological change andserum biochemical disorder were examined and compared between mice with/withoutmTNFR1shRNA plasmid intervention.The expression of mTNFR1 was detected byReal-time PCR,immunohistochemistry staining.The TUNEL method was used todetect hepatocytes apoptosis in MHV-3 induced fulminant hepatitis and then AI(apoptotic index) was evaluated.2.The eukaryotic expression plasmids of human Fas and TNFRⅠgene wereconstructed (pcDNA3.0-hFas and pcDNA3.0-hTNFRⅠ) and have been shownsuccessfully to express hFas and hTNFRⅠprotein,miRNA expression plasmid of hFasand hTNFR1 named p-hFasmiRNA and p-hTNFRⅠmiRNA complimentary to thesequence responsible for hFas and hTNFRⅠrespectively were constructed,meanwhileirrelevant miRNA plasmid was used as control.By respectively cotransfection ofp-hFasmiRNA plus pcDNA3.0-hFas,p-hTNFRⅠmiRNA pluspcDNA3.0-hTNFRⅠexpression construct into 293T cells,the inhibition of hFas andhTNFRⅠexpression was analyzed by real time PCR and western blot.【RESULTS】1.The mTNFR1shRNA plasmid and irrelative shRNA plasmid were successfullyconstructed as evidenced by the restriction enzyme mapping as shown and furtherconfirmed by sequence analysis.The inhibitory effect of mTNFR1 expression bymTNFR1shRNA plasmid was observed in CHO cells.By hydrodynamic delivery,mTNFR1shRNA plasmid significantly reduced mTNFR1 expression in vivo,markedlyameliorates inflammatory infiltration,hepatocytes apoptosis,prolonged the survivaltime period and elevated the survival rate from 0 up to 13.3% in Balb/cJ mice with MHV-3 induced fulminant hepatitis.2.The experiments showed the significant inhibitory effect of p-hFasmiRNA on hFas andp-hTNFRⅠmiRNA on hTNFRⅠexpression at 48h post-transfection both at RNA leveland at protein level.【CONCLUSION】1.Efficient and specific mTNFR1 gene silence targeted by the constructedmTNFR1shRNA plasmid sheds light on the future investigation of gene therapeuticstrategies for patients with fulminant viral hepatitis and disease such as tumor,Metabolic disease,which TNFR1 gene has been shown to play a key role in the diseasedevelopment.In this study we have successfully established the fulminant viral hepatitismodel induced by MHV-3 and demonstrated that hydrodynamic tail vein injectionsefficiently introduced target gene into mice liver,mTNFR1shRNA plasmidsignificantly reduced mTNFR1 expression in vivo,markedly ameliorates inflammatoryinfiltration,hepatocytes apoptosis,prolonged the survival time period and elevated thesurvival rate.2.The study demonstrated that the construct of p-hfgl2miRNA,p-hFasmiRNA andp-hTNFRⅠmiRNA successfully interfered hfgl2,hFas and hTNFRⅠexpression invitro and these provide the foundation for further investigation of these constructs’application in vivo and further more as a therapeutic strategy for a targeting interventionin the disease control to which the gene fgl2,Fas and TNFRⅠcontributed.更多还原