乙型肝炎病毒感染人胎盘绒毛膜癌BeWo细胞体外培养模型的建立

摘要

目的 建立乙型肝炎病毒(HBV)感染人绒毛膜癌滋养层细胞(BeWo)体外培养模型,探讨HBV官内感染机制.方法 应用高病毒载量的HBV阳性血清感染BeWo细胞并传代,应用实时荧光定量聚合酶链反应( PCR)技术检测感染初代细胞和传代细胞内以及上清液中HBV DNA量；应用化学发光微粒子免疫法检测感染后初代细胞和传代细胞的上清液中乙型肝炎表面抗原(HBsAg)、乙型肝炎e抗原(HBeAg)量；应用SABC免疫组化检测感染细胞内HBsAg、乙型肝炎核心抗原(HBcAg)的表达.结果 感染初代各时间点细胞内和上清液中HBVDNA量随时间延长而增加,120 h HBV DNA量最高；传代细胞内和上清液中HBV DNA量随传代次数增加逐渐降低,5代后检测均为阴性.感染初代各时间点细胞的上清液中HBsAg量随时间延长而增加；传代细胞的上清液中HBsAg量在1～3代为阳性,但量逐渐降低,4代后均为阴性.感染初代各时间点细胞和传代细胞的上清液中HBeAg量均为阴性；1～3代细胞HBsAg、HBcAg染色可见阳性细胞.结论 HBV可以感染BeWo细胞,且能在传代细胞中表达；BeWo细胞可用于HBV宫内感染机制的研究.%Objective To establish the in vitro culture model of hepatitis B virus-infected human choriocarcinoma BeWo cells, and evaluate the mechanism of HBV intrauterine infection. Methods BeWo cells were infected with the serum containing high level of HBV, HBV DNA load in the intracellular and supernatant were detected by real-time PCR; supernatant HBsAg and HBeAg quantitative assay was performed by chemiluminescent microparticle immu-noassay; expression of HBsAg and HBcAg in the infected cells was detected with SABC immunohistochemistry. Results In the first passage of infected BeWo cells, the intracellular and supernatant HBV DNA load increased with time, and reached peak 120 hours after infection; In the subsequent passages, the intracellular and supernatant HBV DNA load decreased gradually and became negative after the fifth passage. The supernatant HBsAg quantity in the first passage increased with time, in the first-third passages were all tested as positive, but decreased gradually, all were negative after the fourth passage. The supernatant HBeAg quantity was negative in primary and subsequent passages; The dyed HBsAg and HBcAg in the first-third passage cells showed positive. Conclusion HBV can infect BeWo cells and express in subsequent passages, BeWo cells can be used to study the mechanism of HBV intrauterine infection.