HBV体外感染人胎肝细胞的鉴定方法研究

目的通过HBV体外感染22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HBV DNA,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HBV cccDNA自第8～9天出现阳性.结论HBV在原代培养人22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.

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

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.