原代培养人胎肝细胞体外感染HBV的研究

　　摘　要：目的建立HBV感染人胎肝细胞体外培养系统。方法首先分离、培养人胎肝细胞，然后应用HBV阳性血清感染体外培养的人胎肝细胞；每隔2d收集上清液和肝细胞，应用ELISA,免疫组化法、原位杂交法和斑点杂交法检测上清液和细胞中HBsAg和HBV dNA。结果 上清液中HBsAg在感染后2d～20d均可测出，以感染后4d～16d达高峰(A值在0．22左右)。免疫组化检测细胞中HBsAg呈阳性表达，原位杂交和斑点杂交检测细胞和上清液中HBV dNA也呈阳性表达。结论 HBV在原代培养人胎肝细胞中能稳定复制和表达至少达16d。

Primary human fetal hepatocytes with HBV infection in vitro

Ye Gui Jiang ,Oi Fen Li,Oing Mao ,Yu Ming Wang(Centre of lnfectious Disaases,Southwest Hospital,Third Military Medical university,Chongqing 400038,China)

　　Abstract：AIM to establish a culture systmn of HBV-infected laznan fetal hepatocyte in vitro.METHODS Human fetal hepatocytes were isolated and cultured, then infected with HBV positive serum in vitro.Supematant and cells were collected every 2 days. HbsAg and HBV DNA were detected by ELISA, immunohist-ochemistry, in situ ybridization and dot blot hybridization in cells and supematant.RESULTS HBsAg could be detected from day 2 to day 20 after HBV-infection in supematant and hepatocytes.Secretion of HBsAg reached submit from day 4 to 16 after hBV-infection in supematant (A value: abcut 0.22).HBsAg could be detacted by immunohemistry incells. HBV DNA could be detected by in situ hybridization and dot blot hybridization in cells and supernatont.CONCLUSION Primary human fetal hepatocytes are cumpetont for infectlon with HBV. HBV can stably replicate and express In HBV-infected human fetal hepatocytes,and at least this replication and expression can last 16 days.

　　Keywords：hepatitis b virus; HBV infection; human fetal hepatocyte; cell culture

　　0引言目前，有关HBV的研究，主要是利用HBV基因转染细胞株，但这些细胞株不能完全模拟人体肝细胞的生物学功能，用于研究HBV时，不能完全代表体内情况。用人胎肝细胞作为感染对象，建立HBV感染人胎肝细胞培养系统，可解决上述不足，我们应用HBV感染人胎肝细胞，初步建立了HBV感染人胎肝细胞体外培养系统。1材料和方法1．1材料人工流产22周龄儿肝脏，经体外两步灌流法分离肝细胞，台盼蓝染色人胎肝细胞成活率达90％以上．HBV感染血清取自本科一慢性乙型肝炎患者，ELISA检测HBsAg(+)，HBeAg(+)，抗—HBc(+)，血清斑点杂交HBVDNA(+)，甲、丙、丁、戊、庚型肝炎病毒标志均为阴性。1．2方法采用王宇明etal设计的体外两步灌注法首先分离出总肝脏细胞悬液，再以差速离心方法分离、纯化肝细胞。将分离的人胎肝细胞用含100mL／L小牛血清(FBS)的RPMI1640培养液调整细胞密度为4x108/L，接种于24孔塑料培养板内．每孔含培

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