HBV体外感染人胎肝细胞的鉴定方法研究  

Assays for HBV infection in primary human fetal hepatocytes cultured in vitro

作　　者：王方[1] 王宇明[1] 汤勃[1] 刘俊[1] 刘国栋[1] 王小红[1] 

机构地区：[1]第三军医大学西南医院全军传染病研究所,重庆400038

出　　处：《第三军医大学学报》2004年第1期74-77,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金资助项目 ( 30 170 84 7)~~

摘　　要：目的　通过HBV体外感染 2 2周龄原代培养人胎肝细胞 ,比较常用检测指标的敏感性和特异性。方法　利用HBV阳性血清感染体外培养的原代人胎肝细胞。应用ELISA法检测培养上清中的HBsAg和HBeAg ,免疫组化法检测细胞核中的HBcAg ,荧光定量PCR法检测上清和细胞中HBVDNA ,巢式PCR法检测细胞中cccDNA。结果　胎肝细胞核中的HBcAg于感染后第 2天出现阳性 ,培养上清和细胞中HBVDNA定量自第 4天起检出 ,上清中HBsAg和HBeAg于第 5～ 6天出现阳性 ;细胞中HBVcccDNA自第 8～ 9天出现阳性。结论　HBV在原代培养人 2 2周龄的胎肝细胞中能够稳定复制和表达 ,各种检测指标的灵敏性和特异性不一 ,免疫组化法检测细胞核中的HBcAg敏感性好 ,特异性可。Objective To infect primary cultured human fetal hepatocytes with hepatitis B virus (HBV) in vitro in order to compare the sensitivity and specificity of the conventional assays for infection. Methods The primary cultured 22 week human fetal hepatocytes were incubated with HBV positive serum in vitro . HBsAg and HBeAg in the supernatant were detected by ELISA, HBcAg in nuclei by immunohistochemistry, HBV DNA in supernatant and cells by quantitative fluorescence PCR, and cccDNA in cultured cells by nest PCR. Results HBcAg in nuclei were positive on day 2 after incubation. HBV DNA in supernatants and cells was detected on day 4. HBsAg and HBeAg in supernatants were positive on day 5-6, and cccDNA was positive in cells on day 8-9. Conclusion Stable replication and expression of HBV are observed in HBV infected 22 week human fetal hepatocytes. The sensitivity and specificity of the assays are different. Immunohistochemistry for the detection of HBcAg in nuclei is sensitive and specific, and can be regarded as an easy and convenient method for the detection of HBV infection at the early stage.

关 键 词：乙型肝炎病毒 HBV感染 人胎肝细胞 细胞培养 

分 类 号：R322.47[医药卫生—人体解剖和组织胚胎学] R373.21[医药卫生—基础医学]