HCV Infection In Primary Tupaia Hepatocytes

Hepatitis c is a viral hepatitis caused by hepatitis c virus(HCV)infection.According to data from World Health Organization(WHO),about 170-200 million people(3%of the world's population)worldwide' infected with HCV.The chronic pathogenic progress is the characteristic feature for HCV infection.75%?85%of HCV patients become chronic carriers and a few chronic carriers(approximately 5%)may develop into liver cirrhosis and hepatocellular carcinoma(HCC).,Human and Chimpanzee are the natural host for HCV infection.Because in vitro cell culture models and small animal models with HCV infection are lack,it is limited to study the pathogenic mechanisms of HCV infection..Tree shrew(Tupaia belangeri chinensis)owns essential features as suitable animal models for human disorders:small habitus,rapid propagation,close affinity to primates.In recent years,many studies showed that tree shrew could be infected by HCV and be potential HCV animal model.Culturing the primary liver cells is one of the most important factors to estiblish in vitro cell culture model,but this method is not consummate and needs further optimizition.In addition,another important factor is the interaction between HCV and receptor of host,which could determine HCV infected ability to special hosts and/or special tissues.To study receptors of host will help us to reveal the pathogenic mechanisms of HCV infection and provide theoretical basis for exploring vaccine and drug in clinic.In this study,we firstly isolated primary liver cell of tree shrew by using two-step perfusion method and optimized the method.We compared three kinds of perfusate(Hepes,D-Hank's,and PBS buffer)and the cell count in different centrifugated speed(range from 600 rpm to 1200 rpm),and found that cell activity is best under enviroment with D-Hank's and gradient centrifugation(decreasing from 1000 rpm to 600 rpm).We observed the growth rate of non-parenchymal cells and compared the alteration of primary liver cells with different additive ingredients(serum,ITS and glucose).The primary liver cells could grow well and long(suvival at 40th days)in the cultrue media with 1-2%FBS,1x ITS,and 1%glucose.MTT and EDU dyeing were performed to determine the cell proliferation,and the results showed the count and activity of primary liver cells are better in the most optimal condition than other conditions.This optimized method and condition provides basis for HCV infection to the primary liver cells of tree shrew.Pprimary liver cells were infected by HCV which viral titre is 107 ffu/ml.1.5 was determined to be the best multiplicity of infection(MOI)by MTT and used in the future assays.HCV RNAs in the supernatant were detected by using nested polymerase chain reaction(PCR)and quantitative PCR.The results showed the viral titre could reach 6×104 copies/mL.In order to identify the HCV infection,the proteins were isolated from the primary liver cells and expression of HCV proteins were checked by using Western Blot.All the results indicated HCV could infect the primary liver cells of tree shrew and replicate,but efficiency of infection varied with different HCV batches and host.Finally,we cloned the CD81 gene,which length is 709 bp,into PIRES2 EGF plasmid,and transfected the recombinant plasmid into primary liver cells of tree shrew.We successfully obeserved the green fluorescent proteins by using inverted fluorescence microscope and detected the expression of CD81 at 48th hours after transfection.The results showed that the protein expression level upregulated 5 times in the transfected cells.Primary liver cells transfected with CD81-EGF plasmid were infected by HCV again,and the MOI is still 1.5.These results suggested protein CD81 could increase the HCV infected level to primary liver cells of tree shrew.To sum up,we firstly used and optimized two-step perfusion method to isolate the primary liver cells of tree shrews.Base on the optimized method,we detected the viral titre and expression of HCV proteins by real-time PCR and Western Blot,and identifed HCV could infect primary liver cells of tree shrew.High viral titre was determined in the primary liver cells which transfected with in vitro CD81 protein.We can confirm that protein CD81 played important role in HCV infection.