中文摘要：

目的建立HBV体外感染颗粒细胞模型,研究HBV在颗粒细胞中的复制情况,为深入研究HBV经卵细胞母婴垂直传播提供研究平台。方法原代颗粒细胞体外培养后用HBV阳性血清感染。收集培养上清,在不同时点检测HBsAg、HBeAg定量,实时定量PCR检测HBVDNA。免疫组化检测培养细胞中的HBsAg和HBcAg。巢式PCR检测细胞中的HBVDNA及HBV-mRNA。原位杂交检测细胞内的HBVDNA。结果成功建立了HBV体外感染颗粒细胞模型,在培养上清中可以持续96h检测到HBsAg和HBV DNA,在细胞内检测到HBsAg和HBcAg的阳性信号,PCR扩增显示细胞内有HBVD-NA及HBV-mRNA的存在,原位杂交证实细胞内HBVDNA阳性。结论 HBV能够在体外感染颗粒细胞,并在其内复制,该结果为深入研究HBV经卵细胞传播机制提供了很好的研究平台。

英文摘要：

Objective： By co - cultivation of human GCs with HBV - positive serum, to investigate the infection and replication of hepatitis B virus （HBV） in primary cultured human granulosa ceils. And established a system to study HBV vertical transmission by ovum. Methods ： Human granulosa cells were cultured and infected with HBV - positive serum. Media were collected and assayed for HBsAg and HBeAg by ELISA, and HBV DNA by quantitative PCR. HBsAg and HBeAg were detected by immunoeytoehemistry in cultured cells. Intracellnlar I-IBV DNA and RNA were extracted and amplified by nested PCR. lntraeellular HBV DNA was localized by in situ hybridization. Results： HBsAg in medium could be detected from 4 to 96 h, and HBV DNA could be detected from 12 to 96 h af- ter exposure. HBsAg and HBcAg showed positive signals by immunocytochemistry in cells. HBV DNA and RNA of HBV were ampli- fied successfully by nested PCR. HBV DNA was detected in GC nuclei by in situ hybridization. Conclusion： HBV can infect and replicate in human primary granulosa cells. This culture system could enable us to study vertical transmission of HBV by ovum.