Construction Of The RNAi Expression Plasmids Of Sever Hepatitis-related Genes And Their Biology Effects In Vitro And In Vivo

【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.