Study On Primary Tupaia Hepatocytes: Isolation, Culture, And Infection With HCV

Hepatitis C Virus,the pathogen of hepatitis C,prevails worldwide and endangers the health of human beings seriously.It is estimated that 170～200 millions(about 3%)were in the world,and up to 40 millions(about 2～3%)individuals in China,were infected with HCV.The major drawback on HCV research is the lacking of an efficient HCV culture system and suitable small-animal model.This hampers us to understand the pathogenic characters of virus and chronic disease mechanism.It is also an impediment in developing anti-viral drug and vaccine.Tree shrew(Tupaia belangera),which is a small,easily-breed animal,lives widely in the Southwest of China and Southeast Asia.Tupaiidae has been classified as a species between Insectinora and Primates.The susceptibility of tree shrew with several kinds of medicine virus has been proved.It also been suspected to be a possible animal model of hepatitis C.It was reported that the primary Tupaia Hepatocytes(PTH)could be infected by HCV in several preliminary researches.The confirmation of HCV infection on PTH will be another evidence for the possibility of tree shrew to be an animal model of hepatitis C.It is important to establish PTH isolating,culturing system for HCV infectivity confirmation. Successful culture of PTH also provides an useful tool to understand HCV infection,and to develop vaccine and anti-viral drugs.PTH was isolated with two-step collagenase perfusion method(semi-in situ)from domestic tree shrews,which were captured in the forest of Kunming area,and then cultured successfully.Perfusion was conducted with Hepes solution free of Ca²⁺and Mg²⁺in the direction from portal vein to venae cava inferior,which is the same way with blood cycle, until free of blood cell in liver.The best perfusion velocity was 5-20ml/min,and the optimum concentration of collagenaseâ…£for digestion was 0.025%.It was showed PTH could be dispersed by collagenase digestion at 37℃for about 18min.5×10⁸ with 95%of purity and 90%survive rate cells could been got after washing and centrifuging.It was qualified for cell culture and followed HCV infection.The MEM+5%NCS culture medium with 10ng/ml EGF and 10ng/ml HGF was selected to be mostly suited for long-term culturing of the PTH.In culture progress,PTH developed into a logarithm period after a latent period about 2 days.The time of cell proliferation and growing robustly was from day 3 to day 8.The period of PTH culture could be up to 25 days,by adding medium persistently.The routine method of cell digestion with trypsinase,freezing with FCS-DMSO and resuscitating were used for PTH subculture,but the cell adhibiting and growth was not stable.It is necessary to improve this method.HCVcc supematants derived from plasmid transfected Hela cells was used to infect PTH.The negative strand RNA in infected cells was detected intermittently from day 3 to day 15 after infection,while the positive strand RNA in supernatants was detected intermittently from day 1 to day 18 in suspension by strand specific RT-PCR.And then, PTH was infected with HCVcc,HCVpp with EGFP reporter gene and Luciferase gene. Green fluorescence in HCVpp-EGFP infected cells could be observed by fluorescence microscope.The positive results could be acquired in HCVpp-Luciferase infected cells by luciferase detection.Antigen of HCV in HCVcc infection cells was proved to exist in the infected cells by immuno-fluorescence detection.In summary,the method of PTH isolation and culture was established successfully in this research.The susceptibility of PTH for HCV infection was confirmed preliminary.This could be a useful tool for research on HCV infection mechanism,for anti-viral drug and vaccine development.Meanwhile,this also provides another evidence for tree shrew as hepatitis C animal model.