重型肝炎相关基因的RNA干扰质粒的构建及其体内外干预效应的研究
|
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。
【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.
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。
【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.
引文
1. Hohmann HP, Remy R, BrockhausM, et al. Two different cell types have differentmajor receptors for human tumor necrosis factor ( TNF alpha) [J]. J Biol Chem, 1989, 264 ( 25 ) : 149272-14934.
2. Chan FK, LenardoMJ. A crucial role for p80 TNFR2 in amplifying p60 TNFR1 apoptosis signals in T raves DT. Apoptotic effects of LPS on fibroblasts are indirectly mediated through TNFR1 [J]. J Dent Res, 2004, 83 (9) : 6712676.
3．胡晓，孙晓文，许纯孝．肿瘤坏死因子受体基因转移增强TNF杀伤肿瘤细胞作用的研究[J]．医学研究生学报，2001，14(4)：3432346．
4. Boone E, Vanden BT, van Loo G, et al. Structure /Function analysis of p55 tumor necrosis factor receptor and fas associated death domain. Effect on necrosis in L929sA cells [J]. J Biol
5. Costelli P, Aoki P, Zingaro B, et al. Mice lacking TNFalpha receptors 1 and 2 are resistant to death and fulminant liver injury induced by agonistic anti-Fas antibody [J]. Cell Death Differ,2003, 10 (9) : 99721004.
6. NagakiM, Iwai H, Naiki T, et al. High levels of serum interleukin and tumor necrosis factor alpha are associated with fatality in fulminant hepatitis [J]. J Infect Dis, 2000, 182(4): 11032-1108.
7. Kapadia SB, Brideau-Andersen A, Chisari FV. Interference of hepatitis C virus RNA replication by short interfering RNAs. Proc Natl Acad Sci U S A, 2003, 100: 2014-2018.
8. Castanotto D, Li H, Rossi JJ. Functional siRNA expression from transfected PCR products. RNA, 2002, 8: 1454-1460.
9. Shankar P, Manjunath N, Lieberman J. The prospect of silencing disease using RNA interference.JAMA. 2005,293(11 ): 1367-1373.
10. Barbosa AS, Lin CJ. Gene silencing with RNA interference: a novel tool for the study of physiology and pathophysiology of adrenal cortex Arq Bras Endocrinol Metabol, 2004,48(5):612-619.
11. Stanislawska J,Olszewski WL. RNA interference--significance and applications.Arch Immunol Ther Exp (Warsz), 2005,53(1):39-46.
12. Zaehres H,Lensch MW, Daheron L, et al. High-efficiency RNA interference in human embryonic stem cells.Stem Cells,2005, 23(3): 299-305.
13. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nuc1eotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001, 411 494-498.
14. Kapadia SB, Brideau-Andersen A, Chisari FV. Interference of hepatitis C virus RNA replication by short interfering RNAs. Proc Natl Acad Sci USA, 2003, 100:2014-2018.
15. Schomber T, Kalberer CP, Wodnar-Filipowicz A, et al. Gene silencing by lentivirus-mediated delivery of siRNA in human CD34~+ cells. Blood, 2004, 103:4511-4513.
16. Wydro M, Lehmann P. Green fluorescent protein (GFP)-structure, features and applications in molecular biology of plants. Postepy Biochem, 2004,50(3):282-292.
17. Paillusson A,Hirschi N, Vallan C, et al. A GFP-based reporter system to monitor nonsense-mediated mRNA decay.Nucleic Acids Res, 2005, 33(6):54-57.
18. Reynolds A, Leake D, Boese Q, et al.Rational siRNA design for RNA interference. Nat Biotechnol, 2004, 22(3):326-330.
19. Eithan Galun, Evelyn Zeira, Orit Pappo, et al. Liver regeneration indued by a designer human IL-6/sIL-6R fusion protein reverses severe hepatocellular injury. The FASEB Journal, 2000, 14:1979-1987.
20. Arvelo MB, Cooper JT, Longo C, et al. A20 protects mice from D-galactosamine/lipopolysaccharide acute toxic lethal hepatitis. Hepatology, 2002,35(3): 535-543.
21. Song E, Lee SK, Wang J, et al. RNA interference targeting Fas protects mice from fulminant hepatitis.Nat med, 2003;9(3):347-351.
22. Jacque JM, Triques K, Stevenson M. Modulation of HIV-1 replication by RNA interference. Nature, 2002, 418(6896):435-438
23. Kennerdell JR, Carthew RW. Heritable gene silencing in Drosophila using double-stranded RNA. Nat Biotechnol, 2000, 18(8):896-898.
24. Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells.Proc Natl Acad Sci U S A 2002 ,99(9):6047-6052.
1. Ando K, Moriyama T, Guidotti LG, et al. Mechanisms of class I restricted immunopathology. A transgenic mouse model of fulminant hepatitis. J Exp Med, 1993, 178: 1541-1554.
2. Moriyama T, Guilhot S, Klopchin K, et al. Immunobiology and pathogenesis of hepatocellular injury in hepatitis B virus transgenic mice. Science, 1990, 248: 361-364.
3. Ding JW, Ning Q, Liu MF, et al. Fulminant hepatic failure in murine hepatitis virus strain 3 infection: tissue-specific expression of a novel fgl2 prothrombinase. J Virol,1997,71:9223-9230.
4. Ning Q, Brown D, Parodo J, et al. Ribavirin inhibits viral-induced macrophage production of TNF, IL-1, the procoagulant fgl2 prothrombinase and preserves Th1 cytokine production but inhibits Th2 cytokine response. J Immunol, 1998, 160:3487-3493.
5. Levy GA, MacPhee PJ, Fung LS, et al. The effect of mouse heaptitis virus infection on the microcirculation of the liver. Hepatology, 1983, 3: 964-973.
6. Li C, Fung LS, Chung S, et al. Monoclonal antiprothrombinase (3D4.3) prevents mortality from murine hepatitis virus (MHV-3) infection. J Exp Med, 1992, 176(3):689-697.
7. Fingerote RJ, Abecassis M, Phillips MJ, et al. Loss of resistance to murine hepatitis virus strain 3 infection after treatment with corticosteroids is associated with induction of macrophage procoagulant activity. J Virol, 1996, 70(7): 4275-4282.
8. Zhang G, Budker V, Wolff JA. High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA. Hum Gene Ther, 1999, 10(10):1735-1737.
9. Pawlotsky J.M. Molecular diagnosis of viral hepatitis. Gastroenterology. 2002, 122, 1554-1568.
10. Cheng VC, Lo CM, Lau GK. Current issues and treatment of fulminant hepatic failure including transplantation in Hong Kong and the Far East. Semin Liver Dis. 2003,23,239-250.
11. Higuchi H, Gores G.J. Mechanisms of liver injury: an overview. Curr Mol Med. 2003, 3, 483-490.
12. Nakamoto Y, Kaneko S. Mechanisms of viral hepatitis induced liver injury. Curr Mol Med 2003, 3, 537-544.
13. Bradham CA,Plumpe J,Manns MP,et al.Mechanism of hepatic toxicity in TNF induced liver injury [J].Am J Physiol, 1998, 275,G387-392.
14. Nagata s.Apoptosis by desthfaactor[J].Cell,1997,88,355- 365.
15. Vandenabeel P,Declercq W,Beyaert R,et al.Two tumor necrosis
16. factor receptors:struture and function [J].Trends in Cell Biol,1995,5,392- 399.
17. Bass, BL. Double-Stranded RNA as a Template for Gene Silencing. Cell, 2000, 101, 235-238.
18. Dave RS, Pomerantz RJ. RNA interference: on the road to an alternate therapeutic strategy! Rev Med Virol. 2003, 13, 373-385.
19. Stevenson M. Therapeutic potential of RNA interference. N Engl J Med. 2004, 351, 1772-1777.
20. Higuchi H, Gores GJ. Mechanisms of liver injury: an overview. Curr Mol Med. 2003, 3,483-490.
21. Nakamoto Y, Kaneko S. Mechanisms of viral hepatitis induced liver injury. Curr Mol Med 2003, 3, 537-544.
22. Coleman WB. Mechanisms of human hepatocarcinogenesis. Curr Mol Med. 2003, 3, 573-588.
23. Rhoads J. Natural history and epidemiology of hepatitis C. J Assoc Nurses AIDS Care 14(5 Suppl),2003,18S-25S
1. Kwan-Ho Chung, Christopher C. Hart, et al. Polycistronic RNA polymerase ****expression vectors for RNA interference based on BIC/miR-1550 Nucleic Acids Research, 2006, Vol. 34, No. 7
2. Zhi Ming Zheng, Shuang Tang, et al. Development of resistance to RNAi in mammalian cells. Ann N YAcadSci. 2005 November;1058: 105-118.
3. Hitomi Kimura, Hiroaki Kawasaki, et al. Mouse microRNA-23b regulates expression of Hesl gene in P19 cellso Nucleic Acids Symposium Series No. 48 213-214.
4. Shinya Omoto,Yoichi R. et al. Regulation of human immunodeficiency virus 1 transcription by nef microRNA. Journal of General Virology (2005), 86, 751-755.
5. Vale' rie Plaisance, Amar Abderrahmani, et al. MicroRNA-9 Controls the Expression of Granuphilin/Slp4 and the Secretory Response of Insulin-producing Cells. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 37, pp. 26932-26942,September 15, 2006.
6. Kum-Joo Shin, Estelle A. Wall, et al. A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression. PNAS September 12, 2006 vol. 103 no. 37 13759-13764.
7. Jittima Piriyapongsa, I. King Jordan, et al. A Family of Human MicroRNA Genes from Miniature Inverted-Repeat Transposable Elements. PLoS ONE 2(2):e203. doi:10.1371,February 2007, Issue 2 , e203.
8. RAMESH S. PILLAI. et al. MicroRNA function: Multiple mechanisms for a tiny RNA? RNA (2005), 11:1753-1761. Published by Cold Spring Harbor Laboratory Press.
9. Ines Alvarez-Garcia and Eric A. MiskaMicroRNA functions in animal development and human diseaseDevelopment 132, 4653-4662 Published by The Company of Biologists 2005 doi:10.1242/dev.02073
10. Daniel Boden, Oliver Pusch, Rebecca Silbermann, et al. Enhanced gene silencing of HIV-1 speci(?)c siRNA using microRNA designed hairpins 1154±1158 Nucleic Acids Research, 2004, Vol. 32, No. 3.
11. Xu-Gang Xial, Hongxia Zhou, et al. Pol Ⅱ-Expressed shRNA Knocks Down Sod2 Gene Expression and Causes Phenotypes of the Gene Knockout in Mice. PLoS Genetics, January 2006, Volume 2 .
12. Mario Stevenson, Ph.D. Therapeutic Potential of RNA Interference. N Engl J Med 2004;351:1772-7.
13. Triboulet R, Mari B, Lin YL, et al. Suppression of microRNA-silencing pathway by HIV-1 during virus replication. Science ,2007 Mar 16;315(5818): 1579-82. Epub 2007 Feb
1. Chatterjee S, Johnson PR, Wong KK Jr.Dual-target inhibition of HIV-1 in vitro by means of an adeno-associated virus antisense vector. Science. 1992 Nov 27;258(5087): 1485-8.
2． 李勇年，于敏，等．反义RNA体外抑制丙型肝炎病毒基因表达的研究．Chinese J Exp Clin Virol, December 2004,Vol1 8,No.4
3. Gilboa E, Smith C. Gene therapy for infectious diseases: the AIDS model. Trends Genet. 1994 Apr; 10(4): 139-44. Review.
4. Wakita T, Moradpour D, Tokushihge K, et al. Antiviral effects of antisense RNA on hepatitis C virus RNA translation and expression. J Med Virol, 1999, 57.. 217-222.
5. MaCaffrey AP, et al. Inhibition of hepatitis B virus in mice by RNA interference. Nat Biotechnol, 2003, 21 : 639-644.
6. Shlomai A, Shaul Y. Inhibition of hepatitis B virus expression and replication by RNA interference, Hepotology, 2003, 37, 764-770.
7. Hamasaki K, Nakao K, Matsumoto K, et al. Short interference RNA-directed inhibition of hepatitis B virus replication. FEBS Lett, 2003, 543.. 51-54.
8. MaCaffrey AP, Meuse L, Pham TT, et al. RNA interference in adult mice. Nature, 2002, 418: 38-39.
9. Seo MY, Abrinani S, Houghton M, et al. J Viro, 2003; 77.. 810-812.
10. Welch P J, Tritz R, Yei S, et al. Intracellar application of hairpin ribozyme against hepatitis B virus[J]. Gene Ther, 1997, 4(7).: 736-743.
11. Tan TM, Zhou L, et al. Intracellular inhibition of hepatitis B virus S gene expression by chimeric DNA-RNA phosphorothioate minimized ribozyme. Antisense Nucleic Acid Drug Dev. 2002 Aug; 12(4):257-64.
12. Feng Y, Kong YY, Wang Y, Qi GR. Intracellular inhibition of the replication of hepatitis B virus by hammerhead ribozymes. J Gastroenterol Hepatol. 2001 Oct; 16(10): 1125-30.
13. Feng Y, Kong YY, Wang Y, Qi GR. Inhibition of hepatitis B virus by hammerhead ribozyme targeted to the poly(A) signal sequence in cultured cells. Biol Chem. 2001 Apr;382(4):655-60.
14. Ryu KJ, Kim JH, Lee SW. Ribozyme-mediated selective induction of new gene activity in hepatitis C virus internal ribosome entry site-expressing cells by targeted trans-splicing. Mol Ther. 2003 Mar;7(3):386-95.
15. Sarver N, Cantin EM, Chang PS, Zaia JA, Ladne PA, Stephens DA, Rossi JJ. Ribozymes as potential anti-HIV-1 therapeutic agents. Science. 1990 Mar 9;247(4947): 1222-5.
16. Bunnell BA, Morgan RA. Gene therapy for infectious diseases. Clin Microbiol Rev. 1998 Jan; 11 (1):42-56. Review.
17. Zhang J, Yamada O, Sakamoto T, Yoshida H, Araki H, Murata T, Shimotohno K. Inhibition of hepatitis C virus replication by pol Ⅲ-directed overexpression of RNA decoys corresponding to stem-loop structures in the NS5B coding region. Virology. 2005 Aug 31 ;
18．成军．传染病的基因治疗．肝脏，2001，4(6)：23-31．
19. Proietti E, Bracci L, Puzelli S, Di Pucchio T, Sestili P, De Vincenzi E, Venditti M, Capone I, Seif I. Type I IFN as a natural adjuvant for a protective immune response: lessons from the influenza vaccine model. J Immunol. 2002 Jul 1;169(1):375-83.
20. Patel SD, Moskalenko M, Smith D et al. Impact of chimeric immune receptor extracellar protein domains on T cell function[J]. Gene Ther, 1999, 6.. 412-419.
21．关敏，常雅萍．细胞内抗体抗HIV研究进展．国外医学免疫学分册，2000，24(4)： 208-212．
22. Guilhot S, Guidotti LG, Chisari FV. Interleukin-2 downregulates hepatitis B virus gene expression in transgenic mice by a posttranscriptional mechanism. J Virol. 1993 Dec;67(12):7444-9.
23. Marasco WA, Haseltine WA, Chen SY. Design, intracellular expression, and activity of ahuman anti-human immunodeficiency virus type 1 gp120 single-chain antibody. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7889-93.
24. Huygen K. DNA vaccines: application to tuberculosis. Int J Tuberc Lung Dis. 1998 Dec;2(12):971-8. Review.
25. Brave A, Ljungberg K, Boberg A, Rollman E, et al. Reduced cellular immune responses following immunization with a multi-gene HIV-1 vaccine. Vaccine. 2005 Sep 17
26. Nemchinov LG, Liang TJ, Rifaat MM, et al. Development of a plant-derived subunit vaccine candidate against hepatitis C virus. [J].Arch Virol, 2000, 145: 2557.
27. Uno-Furuta S, Matsuo K, Tamaki S, et al. Immunization with recombinant BCG-HCV elicits HCV-specific cytotoxic T lymphocyte in mice. [J].Vaccine, 2003, 21: 3149.
2. Chan FK, LenardoMJ. A crucial role for p80 TNFR2 in amplifying p60 TNFR1 apoptosis signals in T raves DT. Apoptotic effects of LPS on fibroblasts are indirectly mediated through TNFR1 [J]. J Dent Res, 2004, 83 (9) : 6712676.
3．胡晓，孙晓文，许纯孝．肿瘤坏死因子受体基因转移增强TNF杀伤肿瘤细胞作用的研究[J]．医学研究生学报，2001，14(4)：3432346．
4. Boone E, Vanden BT, van Loo G, et al. Structure /Function analysis of p55 tumor necrosis factor receptor and fas associated death domain. Effect on necrosis in L929sA cells [J]. J Biol
5. Costelli P, Aoki P, Zingaro B, et al. Mice lacking TNFalpha receptors 1 and 2 are resistant to death and fulminant liver injury induced by agonistic anti-Fas antibody [J]. Cell Death Differ,2003, 10 (9) : 99721004.
6. NagakiM, Iwai H, Naiki T, et al. High levels of serum interleukin and tumor necrosis factor alpha are associated with fatality in fulminant hepatitis [J]. J Infect Dis, 2000, 182(4): 11032-1108.
7. Kapadia SB, Brideau-Andersen A, Chisari FV. Interference of hepatitis C virus RNA replication by short interfering RNAs. Proc Natl Acad Sci U S A, 2003, 100: 2014-2018.
8. Castanotto D, Li H, Rossi JJ. Functional siRNA expression from transfected PCR products. RNA, 2002, 8: 1454-1460.
9. Shankar P, Manjunath N, Lieberman J. The prospect of silencing disease using RNA interference.JAMA. 2005,293(11 ): 1367-1373.
10. Barbosa AS, Lin CJ. Gene silencing with RNA interference: a novel tool for the study of physiology and pathophysiology of adrenal cortex Arq Bras Endocrinol Metabol, 2004,48(5):612-619.
11. Stanislawska J,Olszewski WL. RNA interference--significance and applications.Arch Immunol Ther Exp (Warsz), 2005,53(1):39-46.
12. Zaehres H,Lensch MW, Daheron L, et al. High-efficiency RNA interference in human embryonic stem cells.Stem Cells,2005, 23(3): 299-305.
13. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nuc1eotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001, 411 494-498.
14. Kapadia SB, Brideau-Andersen A, Chisari FV. Interference of hepatitis C virus RNA replication by short interfering RNAs. Proc Natl Acad Sci USA, 2003, 100:2014-2018.
15. Schomber T, Kalberer CP, Wodnar-Filipowicz A, et al. Gene silencing by lentivirus-mediated delivery of siRNA in human CD34~+ cells. Blood, 2004, 103:4511-4513.
16. Wydro M, Lehmann P. Green fluorescent protein (GFP)-structure, features and applications in molecular biology of plants. Postepy Biochem, 2004,50(3):282-292.
17. Paillusson A,Hirschi N, Vallan C, et al. A GFP-based reporter system to monitor nonsense-mediated mRNA decay.Nucleic Acids Res, 2005, 33(6):54-57.
18. Reynolds A, Leake D, Boese Q, et al.Rational siRNA design for RNA interference. Nat Biotechnol, 2004, 22(3):326-330.
19. Eithan Galun, Evelyn Zeira, Orit Pappo, et al. Liver regeneration indued by a designer human IL-6/sIL-6R fusion protein reverses severe hepatocellular injury. The FASEB Journal, 2000, 14:1979-1987.
20. Arvelo MB, Cooper JT, Longo C, et al. A20 protects mice from D-galactosamine/lipopolysaccharide acute toxic lethal hepatitis. Hepatology, 2002,35(3): 535-543.
21. Song E, Lee SK, Wang J, et al. RNA interference targeting Fas protects mice from fulminant hepatitis.Nat med, 2003;9(3):347-351.
22. Jacque JM, Triques K, Stevenson M. Modulation of HIV-1 replication by RNA interference. Nature, 2002, 418(6896):435-438
23. Kennerdell JR, Carthew RW. Heritable gene silencing in Drosophila using double-stranded RNA. Nat Biotechnol, 2000, 18(8):896-898.
24. Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells.Proc Natl Acad Sci U S A 2002 ,99(9):6047-6052.
1. Ando K, Moriyama T, Guidotti LG, et al. Mechanisms of class I restricted immunopathology. A transgenic mouse model of fulminant hepatitis. J Exp Med, 1993, 178: 1541-1554.
2. Moriyama T, Guilhot S, Klopchin K, et al. Immunobiology and pathogenesis of hepatocellular injury in hepatitis B virus transgenic mice. Science, 1990, 248: 361-364.
3. Ding JW, Ning Q, Liu MF, et al. Fulminant hepatic failure in murine hepatitis virus strain 3 infection: tissue-specific expression of a novel fgl2 prothrombinase. J Virol,1997,71:9223-9230.
4. Ning Q, Brown D, Parodo J, et al. Ribavirin inhibits viral-induced macrophage production of TNF, IL-1, the procoagulant fgl2 prothrombinase and preserves Th1 cytokine production but inhibits Th2 cytokine response. J Immunol, 1998, 160:3487-3493.
5. Levy GA, MacPhee PJ, Fung LS, et al. The effect of mouse heaptitis virus infection on the microcirculation of the liver. Hepatology, 1983, 3: 964-973.
6. Li C, Fung LS, Chung S, et al. Monoclonal antiprothrombinase (3D4.3) prevents mortality from murine hepatitis virus (MHV-3) infection. J Exp Med, 1992, 176(3):689-697.
7. Fingerote RJ, Abecassis M, Phillips MJ, et al. Loss of resistance to murine hepatitis virus strain 3 infection after treatment with corticosteroids is associated with induction of macrophage procoagulant activity. J Virol, 1996, 70(7): 4275-4282.
8. Zhang G, Budker V, Wolff JA. High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA. Hum Gene Ther, 1999, 10(10):1735-1737.
9. Pawlotsky J.M. Molecular diagnosis of viral hepatitis. Gastroenterology. 2002, 122, 1554-1568.
10. Cheng VC, Lo CM, Lau GK. Current issues and treatment of fulminant hepatic failure including transplantation in Hong Kong and the Far East. Semin Liver Dis. 2003,23,239-250.
11. Higuchi H, Gores G.J. Mechanisms of liver injury: an overview. Curr Mol Med. 2003, 3, 483-490.
12. Nakamoto Y, Kaneko S. Mechanisms of viral hepatitis induced liver injury. Curr Mol Med 2003, 3, 537-544.
13. Bradham CA,Plumpe J,Manns MP,et al.Mechanism of hepatic toxicity in TNF induced liver injury [J].Am J Physiol, 1998, 275,G387-392.
14. Nagata s.Apoptosis by desthfaactor[J].Cell,1997,88,355- 365.
15. Vandenabeel P,Declercq W,Beyaert R,et al.Two tumor necrosis
16. factor receptors:struture and function [J].Trends in Cell Biol,1995,5,392- 399.
17. Bass, BL. Double-Stranded RNA as a Template for Gene Silencing. Cell, 2000, 101, 235-238.
18. Dave RS, Pomerantz RJ. RNA interference: on the road to an alternate therapeutic strategy! Rev Med Virol. 2003, 13, 373-385.
19. Stevenson M. Therapeutic potential of RNA interference. N Engl J Med. 2004, 351, 1772-1777.
20. Higuchi H, Gores GJ. Mechanisms of liver injury: an overview. Curr Mol Med. 2003, 3,483-490.
21. Nakamoto Y, Kaneko S. Mechanisms of viral hepatitis induced liver injury. Curr Mol Med 2003, 3, 537-544.
22. Coleman WB. Mechanisms of human hepatocarcinogenesis. Curr Mol Med. 2003, 3, 573-588.
23. Rhoads J. Natural history and epidemiology of hepatitis C. J Assoc Nurses AIDS Care 14(5 Suppl),2003,18S-25S
1. Kwan-Ho Chung, Christopher C. Hart, et al. Polycistronic RNA polymerase ****expression vectors for RNA interference based on BIC/miR-1550 Nucleic Acids Research, 2006, Vol. 34, No. 7
2. Zhi Ming Zheng, Shuang Tang, et al. Development of resistance to RNAi in mammalian cells. Ann N YAcadSci. 2005 November;1058: 105-118.
3. Hitomi Kimura, Hiroaki Kawasaki, et al. Mouse microRNA-23b regulates expression of Hesl gene in P19 cellso Nucleic Acids Symposium Series No. 48 213-214.
4. Shinya Omoto,Yoichi R. et al. Regulation of human immunodeficiency virus 1 transcription by nef microRNA. Journal of General Virology (2005), 86, 751-755.
5. Vale' rie Plaisance, Amar Abderrahmani, et al. MicroRNA-9 Controls the Expression of Granuphilin/Slp4 and the Secretory Response of Insulin-producing Cells. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 37, pp. 26932-26942,September 15, 2006.
6. Kum-Joo Shin, Estelle A. Wall, et al. A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression. PNAS September 12, 2006 vol. 103 no. 37 13759-13764.
7. Jittima Piriyapongsa, I. King Jordan, et al. A Family of Human MicroRNA Genes from Miniature Inverted-Repeat Transposable Elements. PLoS ONE 2(2):e203. doi:10.1371,February 2007, Issue 2 , e203.
8. RAMESH S. PILLAI. et al. MicroRNA function: Multiple mechanisms for a tiny RNA? RNA (2005), 11:1753-1761. Published by Cold Spring Harbor Laboratory Press.
9. Ines Alvarez-Garcia and Eric A. MiskaMicroRNA functions in animal development and human diseaseDevelopment 132, 4653-4662 Published by The Company of Biologists 2005 doi:10.1242/dev.02073
10. Daniel Boden, Oliver Pusch, Rebecca Silbermann, et al. Enhanced gene silencing of HIV-1 speci(?)c siRNA using microRNA designed hairpins 1154±1158 Nucleic Acids Research, 2004, Vol. 32, No. 3.
11. Xu-Gang Xial, Hongxia Zhou, et al. Pol Ⅱ-Expressed shRNA Knocks Down Sod2 Gene Expression and Causes Phenotypes of the Gene Knockout in Mice. PLoS Genetics, January 2006, Volume 2 .
12. Mario Stevenson, Ph.D. Therapeutic Potential of RNA Interference. N Engl J Med 2004;351:1772-7.
13. Triboulet R, Mari B, Lin YL, et al. Suppression of microRNA-silencing pathway by HIV-1 during virus replication. Science ,2007 Mar 16;315(5818): 1579-82. Epub 2007 Feb
1. Chatterjee S, Johnson PR, Wong KK Jr.Dual-target inhibition of HIV-1 in vitro by means of an adeno-associated virus antisense vector. Science. 1992 Nov 27;258(5087): 1485-8.
2． 李勇年，于敏，等．反义RNA体外抑制丙型肝炎病毒基因表达的研究．Chinese J Exp Clin Virol, December 2004,Vol1 8,No.4
3. Gilboa E, Smith C. Gene therapy for infectious diseases: the AIDS model. Trends Genet. 1994 Apr; 10(4): 139-44. Review.
4. Wakita T, Moradpour D, Tokushihge K, et al. Antiviral effects of antisense RNA on hepatitis C virus RNA translation and expression. J Med Virol, 1999, 57.. 217-222.
5. MaCaffrey AP, et al. Inhibition of hepatitis B virus in mice by RNA interference. Nat Biotechnol, 2003, 21 : 639-644.
6. Shlomai A, Shaul Y. Inhibition of hepatitis B virus expression and replication by RNA interference, Hepotology, 2003, 37, 764-770.
7. Hamasaki K, Nakao K, Matsumoto K, et al. Short interference RNA-directed inhibition of hepatitis B virus replication. FEBS Lett, 2003, 543.. 51-54.
8. MaCaffrey AP, Meuse L, Pham TT, et al. RNA interference in adult mice. Nature, 2002, 418: 38-39.
9. Seo MY, Abrinani S, Houghton M, et al. J Viro, 2003; 77.. 810-812.
10. Welch P J, Tritz R, Yei S, et al. Intracellar application of hairpin ribozyme against hepatitis B virus[J]. Gene Ther, 1997, 4(7).: 736-743.
11. Tan TM, Zhou L, et al. Intracellular inhibition of hepatitis B virus S gene expression by chimeric DNA-RNA phosphorothioate minimized ribozyme. Antisense Nucleic Acid Drug Dev. 2002 Aug; 12(4):257-64.
12. Feng Y, Kong YY, Wang Y, Qi GR. Intracellular inhibition of the replication of hepatitis B virus by hammerhead ribozymes. J Gastroenterol Hepatol. 2001 Oct; 16(10): 1125-30.
13. Feng Y, Kong YY, Wang Y, Qi GR. Inhibition of hepatitis B virus by hammerhead ribozyme targeted to the poly(A) signal sequence in cultured cells. Biol Chem. 2001 Apr;382(4):655-60.
14. Ryu KJ, Kim JH, Lee SW. Ribozyme-mediated selective induction of new gene activity in hepatitis C virus internal ribosome entry site-expressing cells by targeted trans-splicing. Mol Ther. 2003 Mar;7(3):386-95.
15. Sarver N, Cantin EM, Chang PS, Zaia JA, Ladne PA, Stephens DA, Rossi JJ. Ribozymes as potential anti-HIV-1 therapeutic agents. Science. 1990 Mar 9;247(4947): 1222-5.
16. Bunnell BA, Morgan RA. Gene therapy for infectious diseases. Clin Microbiol Rev. 1998 Jan; 11 (1):42-56. Review.
17. Zhang J, Yamada O, Sakamoto T, Yoshida H, Araki H, Murata T, Shimotohno K. Inhibition of hepatitis C virus replication by pol Ⅲ-directed overexpression of RNA decoys corresponding to stem-loop structures in the NS5B coding region. Virology. 2005 Aug 31 ;
18．成军．传染病的基因治疗．肝脏，2001，4(6)：23-31．
19. Proietti E, Bracci L, Puzelli S, Di Pucchio T, Sestili P, De Vincenzi E, Venditti M, Capone I, Seif I. Type I IFN as a natural adjuvant for a protective immune response: lessons from the influenza vaccine model. J Immunol. 2002 Jul 1;169(1):375-83.
20. Patel SD, Moskalenko M, Smith D et al. Impact of chimeric immune receptor extracellar protein domains on T cell function[J]. Gene Ther, 1999, 6.. 412-419.
21．关敏，常雅萍．细胞内抗体抗HIV研究进展．国外医学免疫学分册，2000，24(4)： 208-212．
22. Guilhot S, Guidotti LG, Chisari FV. Interleukin-2 downregulates hepatitis B virus gene expression in transgenic mice by a posttranscriptional mechanism. J Virol. 1993 Dec;67(12):7444-9.
23. Marasco WA, Haseltine WA, Chen SY. Design, intracellular expression, and activity of ahuman anti-human immunodeficiency virus type 1 gp120 single-chain antibody. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7889-93.
24. Huygen K. DNA vaccines: application to tuberculosis. Int J Tuberc Lung Dis. 1998 Dec;2(12):971-8. Review.
25. Brave A, Ljungberg K, Boberg A, Rollman E, et al. Reduced cellular immune responses following immunization with a multi-gene HIV-1 vaccine. Vaccine. 2005 Sep 17
26. Nemchinov LG, Liang TJ, Rifaat MM, et al. Development of a plant-derived subunit vaccine candidate against hepatitis C virus. [J].Arch Virol, 2000, 145: 2557.
27. Uno-Furuta S, Matsuo K, Tamaki S, et al. Immunization with recombinant BCG-HCV elicits HCV-specific cytotoxic T lymphocyte in mice. [J].Vaccine, 2003, 21: 3149.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |