HBV X基因转化人肝细胞的实验研究

Experimental Study on HBV X Gene Transformed Human Hepatocyte

选取已稳定转染HBV X基因的QSG7701细胞(pCMV X/QSG7701),并以稳定转染空质粒pRc/CMV2的QSG7701细胞(pRcCMV2/QSG7701)及未转染的QSG7701细胞为对照,用免疫荧光方法检测pCMV X/QSG7701细胞中HBX蛋白的分布;用RT-PCR方法和免疫细胞化学法分别检测细胞中c-Myc表达;用光镜及电镜观察细胞形态学变化,进一步研究乙肝病毒(HBV)X基因对永生化非瘤性人肝细胞QSG7701的转化作用.研究发现,乙肝病毒X(HBX)蛋白主要分布在细胞膜及细胞浆;c-Myc mRNA及c-Myc蛋白在3种细胞中均有表达,但转染了X基因的细胞中c-Myc的表达水平较其他两组明显升高(P<0.01);光镜和电镜下观察发现pCMV X/QSG细胞的胞核较大,核膜增厚,核仁肥大且增多,核/浆比例失调,可见少量多核巨细胞,pRc-CMV2/QSG7701细胞与QSG7701细胞核浆比正常,染色质分布均匀,胞核大小及形态正常.结果表明,HBVX基因成功转染了QSG7701细胞,HBV X蛋白主要分布于转染细胞的细胞膜和细胞浆中,HBX可能通过上调转染细胞中c-Myc蛋白的表达而使细胞具备恶性化生长的倾向.

The cell transfected with expression plasmid pCMV/X （pCMV X/QSG cell line） as well as cells transfected with control plasmids pRc/CMV2 （pRcCMV2/QSG cell line） and untransfected cell line QSG7701, were recovery and cultured. The expression of HBV X protein was detected by immunofluorescence in pCMV X/QSG7701 cells. The expressions of c-Myc mRNA and protein was detected by semi-quantitative RT-PCR and immunohistochemistry in pCMV X/QSG7701 cells and in cells of control groups, respectively. Cellular morphology were observated by light microscope and electron microscope. It was showed that HBV X protein was located in nuclear membrane and cytoplasm of transfected cells, c-Myc mRNA and protein were expressed in the cells of three groups, however, c-Myc mRNA and protein expression in pCMV X/QSG7701 cells was significantly higher than which in the other two cell groups （P〈 0.01 ）. pCMV X/QSG7701 cells had a bigger cellular nucleus and chromatospherite, the ratio of nucleus and cytoplasm was imbalance; Muhinucleated giant cells could be seen in pCMV X/QSG7701 group. Cellular morphous of pRc/CMV2 QSG7701 and QSG7701 cells were normal, the chromoplasm was uniformly distributed, the proportion of nucleus and cytoplasm was normal. HBV X gene transfected immortalized human liver cell line QSG7701 successfully. HBV X protein was located in nuclear membrane and cytoplasm of the transfected cells. HBV X transform transfected QSG7701 cells might be up-regulate the expression of c-Myc protein.