乙型肝炎病毒感染原代培养人绒毛膜滋养层细胞的实验研究

目的探讨原代培养的人绒毛膜滋养层细胞感染乙型肝炎病毒（hepatitis B virus,HBV）的可能性及规律。方法采用胰酶/DNA酶序贯消化及Percoll密度梯度离心分离纯化人绒毛膜滋养层细胞后,采用血清直接感染法进行滋养层细胞的HBV感染,同时通过与对正常成人肝细胞株L02细胞的HBV感染后及转染HBV的肝癌细胞株（HepG2.2.15）的传代培养比较,实时荧光定量PCR检测培养上清的HBV DNA;并用透射电镜观察HBV感染后滋养层细胞的超微结构改变及其在滋养层细胞中的定位。结果原代培养的滋养层细胞感染HBV后释放至培养上清中的HBV DNA于感染后48-72h达高峰,而L02细胞则于感染后12-24h达高峰,HepG2.2.15细胞株感染后则一直攀升。透射电镜观察HBV感染后滋养层细胞超微结构发生了明显改变,主要表现为溶酶体大量增生,出现空泡状结构,并可观察到病毒样颗粒,主要定位于细胞核周。结论HBV可以感染滋养层细胞,定位于核周并导致滋养层细胞的超微结构改变。

On HBV infection of trophoblastic cells in primary culture

Objective To investigate the possibility and regulation of that hepatitis B virus(HBV)infects trophoblastic cells in primary culture.Methods Trypsin and DNAse sequential digestion and Percoll density centrifugation were used to separate and purify human chorionic trophoblastic cells.The direct serum(HBV DNA positive)infection method was adopted to infect trophoblastic cells.The results of HBV infection of trophoblastic cells were compared with that of normal human adult liver cell line L02 and that of liver cancer cell line HepG2.2.15.Real-time PCR was used to detect HBV DNA in culture supernatant.Further,ultrastructural characteristics of infected trophoblastic cells were observed with transmission electron microscope.Results The HBV DNA in culture supernatant that released by HBV infected trophoblastic cells reached the peak in 48-72 h.For L02 cell,it occurred in 12-24 h and for HepG2.2.15 cell line,it kept increasing.The ultrastructure of infected HBV trophoblastic cells differed obviously from that of normal cells,and showed hyperplasia of lysosome,appearance of vacuoles and virus-like particles around the nucleus.Conclusion Trophoblastic cells could be infected by HBV,and the cellular ultrastructure changed dramatically following HBV infection.