乙型肝炎病毒体外感染人绒毛膜癌JEG3细胞的实验研究

Experimental Study of Human Choriocarcinoma Cell Line JEG3 Infected with Hepatitis B Virus in Vitro

目的建立乙型肝炎病毒(HBV)体外感染人绒毛膜癌滋养层细胞(JEG3)模型,探讨HBV宫内感染机制。方法应用HBV阳性且高载量的血清,感染JEG3细胞。应用实时荧光定量PCR(RT-PCR)技术检测感染后细胞上清液和细胞内的HBV载量;应用ELISA方法检测感染后不同时间、不同代次上清液的HBsAg、HBeAg浓度;应用SP免疫组化检测感染细胞内HBsAg、HBeAg的表达。结果感染初代的JEG3细胞,随着时间增加病毒载量亦增加。传代后细胞及上清中的HBV载量随传代次数增加逐渐降低,5代后检测均为阴性;感染初代上清液HBsAg含量随时间增加,120h含量最高。传代细胞的上清液HBsAg检测1～4代为阳性,但浓度逐渐降低,5代后均为阴性。上清液的HBeAg检测,各时间点、各代均为阴性;1、2、3、4代细胞HBsAg、HBcAg染色可见阳性细胞。结论HBV可以在体外感染滋养层细胞,且能在传代细胞中有持续的表达。感染过程中参与调控的相关细胞因子及HBV复制的持续与否有待进一步探讨。

Objective To establish the human trophoblast cell model with human choriocarcinoma cell line JEG3 infected with hepatitis B virus （HBV） in vitro and explore the mechanism of intrauterine HBV infection. Methods Human choriocarcinoma cell line JEG3 was infected with the serum containing high level of HBV. HBV DNA load in the supematant and the cells were detected by real-time PCR. The levels of HBsAg and HBeAg in the supematant of serial passages were measured by ELISA at different times. The expression of HBsAg and HBcAg in the infected cells were determined by immunohisochemical SP method. Resallts In the first passage of infected JEG3 cells, HBV load increased with time. HBV load decreased after the first passage and disappeared after the frith passage. In the first passage, the content of HBsAg increased with time and reached the peak 120 hours after the infection. HBsAg was positive in the supematant of the first 4 passages, but decreased gradually and became negative after the fifth passage. HBeAg was negative in all passages. Cells infected with HBsAg and HBcAg were detected by immunohisochemical staining in the first 4 passages Conclusion HBV can infect JEG3 in vitro and express in the first 4 passages. The related cytokines during the process of infection and the mechanism of HBV replication need further study.