HBV全基因组1.3倍体HepG2细胞模型的构建与表达

Construction of HBV whole-genome 1. 3 ploid HepG2 cell model and expression of HBV biomarkers

目的构建含HBV全基因组1.3倍体(1.3 ploid HBV,HBV 1.3P)的HepG2细胞模型,并观察HBV生物标志物的表达规律。方法通过腺病毒将全基因组合成的1.3倍HBV序列转染入Hep G2细胞中,构建HBV 1.3P-HepG2细胞模型,采用琼脂糖凝胶电泳鉴定HBV 1.3P重组腺病毒质粒,利用ABI 3730测序仪确定重组腺病毒质粒中含有正确的1.3倍HBV序列,荧光显微镜下鉴定HBV 1.3P腺病毒感染HepG2细胞的最佳感染复数(MOI),qRT-PCR测定0~9 d HBV 1.3P-HepG2细胞模型中HBV DNA、ccc DNA的表达水平,化学发光法、免疫荧光法分别检测上清液及细胞内HBsAg、HBeAg的表达量。计量资料多组间比较采用方差分析,进一步两两比较采用SNK-q检验。结果当MOI=40时,HepG2细胞被HBV 1.3P重组腺病毒感染的效率达到90%以上;在感染第2天即可分别在上清液和细胞内中检测出HBV DNA、ccc DNA、HBsAg及HBeAg等标志物的表达,在4~6 d达到峰值水平,7 d之后逐渐下降。结论所构建的HBV全基因组1.3倍体HepG2细胞模型能稳定表达HBV标志物,为开展HBV的相关研究奠定了基础。

Objective To construct an HepG2 cell model containing HBV whole -genome 1.3 ploid （HBV 1.3P）, and to investigate the expression of HBV biomarkers. Methods HepG2 cells were transfected with the 1.3 - ploid whole - genome HBV DNA sequence using the adenovirus vector to construct an HBV 1.3P - HepG2 cell model. Agarose gel electrophoresis was used to identify HBV 1.3P recombinant adenovirus plasmids, and the ABI 3730 sequencer was used to confirm whether the recombinant adenovirus plasmids had correct HBV 1.3P sequence. The optimal multiplicity of infection （MOI） of HepG2 cells infected by the HBV 1.3P adenovirus was determined under a fluores- cence microscope. Quantitative real - time PCR was used to measure the expression of HBV DNA and eccDNA in the HBV 1.3P - HepG2 cell model at 0 - 9 days, and chemiluminescence and immunofluorescence assay were used to measure the expression of HBsAg and HBeAg, respectively, in supernatant and cells. An analysis of variance was used for comparison of continuous data between multiple groups, and the SNK - q test was used for comparison between any two groups. Results When MOI was 40, the efficiency of HepG2 cells being infected by HBV 1.3P recombinant adenovirus reached above 90%. On the second day of infection, the expression of the biomarkers HBV DNA, cccD- NA, HBsAg, and HBeAg was detected in supernatant and cells, and the levels of these biomarkers reached peak values at 4 - 6 days and gradually decreased after 7 days. Conclusion This HBV 1.3P - HepG2 cell model can express HBV biomarkers stably, which lays a foundation for future research on HBV.