HepG2.2.15细胞模型功能的重新评价:分泌HBVDNA、cccDNA及血清学标志物的动态变化研究

Re-evaluation of the functioning of a HepG2.2.15 cell model: Dynamic changes in HBV DNA, cccDNA, and serological markers secreted by cells

目的研究HepG2.2.15细胞分泌特点及动态变化,建立稳定的抗HBV药物筛选平台。方法采用微量细胞培养法培养HepG2.2.15细胞株,于第3、6、9、12、15d收集培养上清液,采用ELISA方法检测preS1、HBsAg和HbeAg,采用Abbot试剂盒定量检测HBVDNA,采用Roche荧光定量PCR试剂盒检测HBVcccDNA。结果 HepG2.2.15细胞分泌preS1、HBsAg、HBeAg、HBVDNA的量随着细胞培养时间的延长逐渐增加,其中HBsAg在第9d达到分泌高峰,其余均在第12d达到分泌高峰。HBVcccDNA在第9d检测值仍为0,第12d时检测值达24×103copies/ml。结论 HepG2.2.15细胞具有稳定的分泌HBV病毒及相关抗原的生物学功能,是比较理想的病毒复制及抗HBVDNA药物筛选的细胞模型。本研究所用细胞株及实验方法可靠、稳定且重复性好,适用于抗HBV药物筛选等研究。

Objective To establish a stable platform for the evaluation of anti-HBV drugs via timely detection of Pre-S1, HBsAg, HBeAg, HBV DNA and HBV cccDNA in HepG2.2.15 cells. Methods HepG2.2.15 cells were cultured in vitro using microplates. The supernatant was collected on days 3, 6, 9, 12, and 15. Pre-S1, HBsAg, and HBeAg were detected using ELISA, HBV DNA was detected with an Abbot kit for quantitative detection of HBV DNA, and HBV cccDNA was detected with a Roche kit for fluorescence quantitative PCR. Results The amount of Pre-S1, HBsAg, and HBeAg secreted and the amount of HBV DNA gradually increased with the culturing time. HBsAg peaked day 9, while the others peaked on day 12. HBV cccDNA was not detected until day 12, when the determined amount was 24×10^3 copies/ml. Conclusion HepG2.2.15 cells consistently secrete HBV and can serve as an ideal cell model for further study of the replication of HBV and to help screen anti-HBV drugs. In conjunction with the cells used, the methods used in this study were reliable, stable, and highly reproducible, indicating their suitability to screening and evaluation of anti-HBV drugs.