Mediate Function Of Human CD81 And Occludin To HCV Infection On Marrow Mesenchymal Stem Cells From Rhesus Bone

Hepatitis C virus is classified as the member of Flaviviridae family.It was estimeted that more than 200 million population was infected with HCV in worldwide since its discovery in 1989,with the annual infected cases of about 35 thousand.The major transmission routes of HCV are blood transmission,maternal-neonatal transmission and sexual transmission.Once infected,85%of cases will turned as chronic disease,and more tha 20%of them will developed into liver fibrosis,cirrhosis,and 5%of them died as hepatocellular carcinoma.Interferon-alpha/Ribavirin has been widely used for clinical treatment.Moreover,DAA protease inhibitor Boceprevir(Telaprevir)and interferon combined with Ribavirin also showed a better therapeutic effect.Recently,nucleoside RdRp enzyme inhibitor of Sofobuvir and NS5A inhibitor of Velpatasvir has been approved by FDA for clinical using,and Epclusa,a combination of Sovaldi(Sofobuvir)and Velpatasvir,also showed a good curative effect on the patients without cirrhosis or decopensated cirrhosis(mild cirrhosis).However,these treatments were expensive and drug resisitant mutations of HCV were found frequently.Upto date,vaccine for HCV prevention is unavalible,the development of drugs and vaccine is still necessary.As a platform for the development of drugs and vaccines,a suitable animal model play an irreplaceable role.There have some animal models,humanized mice,transgenic mice,rats,tupaia,chimpanzee and so on,were established.Howeve,the high cost,long experimental period,feeding conditions and ethical issues have limitted their using.With the development of genetic engineering techniques,it is possible to establish genetic engineering primate model of hepatitis C.CD81 and tight junction protein(Occludin),the members of transmembrane 4 superfamily,are considered as the candidate receptors of HCV.It was demonstrated that the envelop glycoprotein E2 of HCV could bind with CD81 on cell membrane,which may introduce HCV virion into cell having the positively correlation with expression dose of CD81.Occludin is considered as an important factor affecting the level of HCV infection,it has been shown that overexpression this receptor in human hepatocyte can significantly increase the infection of pseudovirus.To establish the cell model with stable expression od HCV receptors,the genes of human CD81 and Occludin were amplified,and then ligated into the lentiviral vector of pLVX-IRES-Zs Green1 respectively.Subsequently,the reconstructed vecrots were transfected into 293T cells by calcium phosphate method.The titer of obtained lentivirus particles from the harvested supernatant was upto 107TU/mL after ultra centrifugation.Furthermore,this lentivirus was used to infecte human hepatoma HepG2 cells,Chinese hamster ovary(CHO)cells and rhesus monkey bone marrow mesenchymal stem cells BMSCs.The expression of CD81 and Occludin mRNA were detected in HepG2 and CHO cells by indirect immunofluorescence,flow cytometry and western-blot.Furthermore,mesenchymal stem cells(MSC)were isolated from the femur of rhesus monkey.The results of flow cytometry analysis showed that CD34 and CD45 were negative and CD44,CD90 and CD 105 were positive in MSC.In the differentiation experiment,the bone mesenchymal stem cells were cultuered with the potential abilities to differentiate into osteoblasts,adipocytes and chondrocytes after cultured for 7 days and 21 days with the corresponding medium respectively.The staining results with oil red,alizarin red and alcian blue further confirmed that the obtained cells are bone mesenchymal stem cells and have the potential for differentiation.The lentivirus was used to infect BMSCs in the same way and expression of HCV receptors in MSC were tested as well.At last,the MSC with HCV recptors expression were infected with HCVpp and HCVcc.The results of RT-PCR suggested that the expression of CD81 in HepG2 could increase the infectivity of HCV endocytosis in agreement with the experiment of immunofluorescence.However,this findings of Occludin in CHO cells was not obvious.Comparatively,BMSCs infected with HCVpp demonstrate the existence of GFP protein in immunofluoscence.These results have showed that the expression of CD81 and Occludin in MSC could mediate the entry of HCVpp.In summary,mesenchymal stem cells model with HCV reseptors(CD81 and Occludin)expression were successfully constructed,and the infectivity of HCVcc and HCVpp on these MSCs were preliminary confirmed.These findings have provided a theoretical foundation for animal models in vivo,which is also essential for the research on the mechanism of HCV infection and replication.