C反应蛋白基因对HepG2.215细胞株乙型肝炎病毒复制的影响

Effect of CRP Gene on HBV Replication in HepG2.215 Cell Lines

目的探讨C反应蛋白(C-reactive protein,CRP)基因对肝癌HepG2.215细胞株乙型肝炎病毒(HBV)复制的影响。方法用CRP基因过表达载体、CRP基因沉默载体、CRP基因突变载体及CRP基因空载体的慢病毒转染并筛选HepG2.215细胞稳定株进行研究。分为实验组、阴性对照组和空白对照组。采用RT-qPCR检测各组CRPmRNA表达水平,Western blot检测CRP蛋白表达水平,定量PCR检测HBVcccDNA、HBVDNA表达水平,免疫荧光检测乙型肝炎病毒核心抗原(HBcAg)表达情况,透射电镜检查观察HepG2.215细胞病毒复制情况,采用ELISA检测培养上清中HBsAg、HBeAg的表达水平,采用RT-qPCR检测HBVmRNA、HBVpgRNA的表达。结果CRP在HepG2.215细胞中的表达高于HepG2细胞(P<0.05)。CRP基因过表达组与阴性对照组相比较,HepG2.215细胞CRP蛋白和CRPmRNA、HBVcccDNA、HBVpgRNA及HBcAg表达升高,沉默CRP基因上述各项指标表达水平下降(P均<0.05);CRP基因过表达组与空白对照组相比较,HepG2.215细胞HBVmRNA、HBVDNA、HBsAg、HBeAg表达水平升高;CRP基因沉默组与空白对照组相比较,上述各项指标表达均降低(P均<0.05);CRP基因突变表达组与空白对照组相比较,HepG2.215细胞HBsAg表达水平升高(P<0.05)。结论CRP可促进肝癌HepG2.215细胞HBV的复制,并促进HBV相关抗原HBsAg、HBeAg、HBcAg的表达。CRP突变对HBsAg表达有一定促进作用。

Objective The aim of this study was to investigate the effect of CRP gene on HBV replication in hepatocellular carcinoma HepG2.215 cell lines.Methods HepG2.215 cells were transfected with lentivirus containing CRP gene overexpression vector,CRP gene silencing vector,CRP gene mutation vector and CRP gene empty vector respectively,and stable cell lines of HepG2.215 were screened for subsequent experiments,which were divided into experimental groups,negative control groups and blank control groups.the expression of CRPmRNA were detected by RT-qPCR and the CRP protein were detected by Western blot,the expression of HBVcccDNA and HBVDNA were detected by quantitative PCR,the expression of HBcAg was detected by immunofluorescence and transmission electron microscopy(TEM)was used to observe HBV virus replication.The expression of HBsAg and HBeAg in culture supernatants were detected by ELISA.RT-qPCR were used to detect HBVmRNA and HBVpgRNA.Results The expression of CRP in HepG2.215 cells was significantly increased compared with that in HepG2 cells(P<0.05).The overexpression group compared with the negative control group,the expression of CRP protein and CRPmRNA,HBVcccDNA,HBVpgRNA and HBcAg in HepG2.215 cells were significantly increased,and the expression of the above indicators of silencing CRP gene were decreased(P all<0.05).The expression levels of HBVmRNA,HBVDNA,HBsAg and HBeAg in HepG2.215 cells were significantly increased in the CRP gene overexpression group compared with the blank control group,while the expression levels of the above indicators were decreased in the CRP gene silenced group compared with the blank control group(P all<0.05).Compared with the blank control group,the expression level of HBsAg in HepG2.215 cells was increased in CRP gene mutated group(P<0.05).Conclusion CRP can promote HBV replication and the expression of HBsAg,HBeAg and HBcAg in HepG2.215 hepatoma cells.CRP mutation can promote the expression of HBsAg.