丙型肝炎病毒体外感染HepG2细胞模型的建立

Establishment of Hepatitis C Virus Infection Model in vitro by Using Human Hepatoma Cells

目的:建立近似自然感染的丙型肝炎病毒(hepatitisCvirus,HCV)体外感染细胞模型。方法:将含10%HCVRNA阳性血清的接种液和HepG2细胞在37℃、5%CO2条件下共同孵育24h,再加含新生牛血清、地塞米松、胰岛素及硫酸亚铁的维持培养液,置32℃、5%CO2条件下培养,以后每3～4d传代一次,定期收集培养上清液和细胞。应用RT鄄nested鄄PCR、免疫组化及Westernblot进行病毒核酸和蛋白质的检测。结果:RT鄄nested鄄PCR法检测发现在接种病毒后第4～16天的细胞标本中可检测到HCVRNA正链,接种病毒后第4～24天的细胞标本则可检测到HCVRNA副链。通过免疫组化和Westernblot检测,病毒蛋白(NS3)能在第4天检出。结论:HCV体外感染HepG2细胞模型的建立,可望用于HCV吸附肝细胞机制研究及疫苗中和试验。

X Objective:To establish Hepatitis C Virus(HCV)infection cell model in vitro resemble in vivo.Methods:Human hepatoma cells(HepG2)was inoculated with HCV RNA positive serum at37℃,5%CO 2 for24hours.Then bovine calf serum,dexamethasone,insulin and FeSO 4 were added to the medium at32℃,5%CO 2 for cultivation.Cells and super-natant were harvested every3～4days.Nucleic acid and protien of the virus were detected by RT-nested-PCR,immuno-histochemistry and Western blot.Results:Plus-strand HCV RNA was detectable in HepG2cell pellets from4th to16th day after inoculation and minus-strand HCV RNA from4th to24th day.The antigen of virus(NS3)could be detected in cells harvested at the fourth day after inoculation by immunohistochemistry and Western blot.Conclusions:HCV infec-tion cell model in vitro is established and could be used in studying the mechanism of HCV absorpting hepatocyte and vaccinal neutrolization test.