Study On HCV Susceptible Cell Model By Establishing Bone Marrow Mesenchymal Stem Cells Transfected With MiR-122 And CD81/Occludin In Tree Shrew

Hepatitis c virus(HCV)was discovered in 1989,initially termed non-A,non-B hepatitis,which seriously endangers human health.According to the WHO Global Hepatitis Report in 2017,the number of people infected with HCV in the world has reached 710 million,and the number of people infected with HCV is increasing every year.Although the anti-HCV drug-Sofosbuvir has come to the market in 2015,but due to the expensive,it is difficult for ordinary patients.China has long-acting interferon pegbin,which was listed in 2016.Development of more efficient therapies has been hampered by lack of in vivo and in vitro model systems.At present,the main cell models of HCV are lymphocyte,primary hepatocyte model,hepatocellular carcinoma cell line and liver-like cell model derived from induced pluripotent stem cells(iPS);animal models mainly include chimpanzee,mouse and rat model.However,these cells and animal models have their own limitations,thus limiting their applications.Tree shrews are closer to non-human primates than rodents.Mesenchymal stem cells(MSCs)are a heterogeneous subset of stromal stem cells that have the ability of self-renewal and multipotency.Bone marrow mesenchymal stem cell(BM-MSCs)are a kind of MSC derived from bone marrow(BM),allowing robust in vitro culture expansion,which can be transfected variety of gene through viral vector,and express the gene efficiency,meanwhile maintain the characteristics of BM-MSCs unchanged.Compared with rodent BM-MSCs,tree shrew BM-MSCs have irreplaceable advantages in HCV related research.Due to miR-122 not only played an importantrole in differentiation of fetal liv-er stem/progenitor cells into hepatocyte-like cells,but also promoted HCV replication,we transfected miR-122 into BM-MSCs of tree shrew.And transfected CD81 and Occludin receptors,which comprised the minimal human factors required for HCV uptake by rodent cells,into BM-MSCs of tree shrew.The research contents and results are as follows:1.Isolation,culture and identification of bone marrow mesenchymal stem cells from tree shrews.Density gradient centrifugation combined with adherent culture method was used to isolate,amplify and purify bone marrow mesenchymal stem cells from tree shrew in vitro.Morphological observation was carried out by inverted phase contrast microscope.The cells were induced to differentiate into adipogenic,osteoblasts and neuron-like cells.BM-MSCs were identified as the isolated cells.2.Construction of CD81/OCLN/miR-122 lentiviral vector and lentivirus transfection into tree shrew BM-MSCs.Lentiviral vector containing human CD81/OCLN/miR-122 was constructed and transfected into BM-MSCs.The expression of CD81,OCLN and miR-122 genes could be detected by real-time fluorescence quantitative PCR.The expression of CD81 and OCLN molecules could be detected by flow cytometry.The proteins of.CD81 and OCLN could be tested in BM-MSCs of tree shrews by Western-blot.After transfected with human CD81/OCLN lentiviral vector,the differentiation potential of osteogenic and adipocytes is lower than that of untransfected.3.The study on HCV infected bone marrow mesenchymal stem cells of tree shrews.In this part,we used HCVcc to infect OCLN/BM-MSCs,CD81/BM-MSCs,miR-122/BM-MSCs,CD81/miR-122BM-MSCs,OCLN/miR-122/BM-MSCs,OCLN/CD81/BM-MSCs,OCLN/CD81/miR-122/BM-MSCs,the negative-stranded RNA can be detected by nested PCR,which indicated that the cells could support the entry and replication of HCVcc.The supernatants of OCLN/CD81/BM-MSCs and OCLN/CD81/miR-122/BM-MSCs can produce infectious HCV particles.It was confirmed that the CD81 and OCLN receptors was comprised the minimal human factors required for HCV uptake in this cells.And the presence of miR-122 could increase the susceptibility to HCV.What's more,the VEGF may significantly increase the susceptibility of target cells to HCVcc by disrupting the polarity of cells.In conclusion,we established BM-MSCs model of HCV.HCV can complete the life cycle and produce infectious virus particles on this cell.This is the first time to establish a HCV cell model by using BM-MSCs,which provides a new technical platform for HCV life cycle research,anti-HCV drug screening,vaccine development and pathogenesis research.