乙型肝炎病毒X蛋白对肝癌细胞生长的影响

Effect of hepatitis B virus X protein on the growth of liver cancer cells

目的探讨乙型肝炎病毒X蛋白(hepatitis Bvirus Xprotein,HBx)对肝癌细胞恶性程度的影响。方法构建携带HBVx基因真核表达载体pcDNA3/HBx,以脂质体介导转染HepG2肝癌细胞,建立可稳定表达HBx的肝癌细胞系HepG2-HBx细胞,同时设空载体pcDNA3转染细胞HepG2-pcDNA3及未转染HepG2细胞为对照组。PCR法扩增Neo基因检测插入的质粒DNA片段,免疫荧光检测HBx的表达。通过生长曲线测定、平板克隆形成实验、MTT比色实验、Hoechst33342核形态学染色观察及流式细胞仪测定,了解稳定转染细胞的生物学行为变化。结果与对照组相比,转染pcDNA3/HBx的HepG2-HBx细胞生长速度加快,其倍增时间缩短(28h对32.5h或34h,P<0.05),克隆形成率增加[(10.12±0.23)%对(5.33±0.19)%或(5.19±0.28)%,P<0.05],细胞周期分析显示由G0/G1期-S期的进程明显加快。细胞凋亡检测显示HepG2-HBx细胞可抵抗放线菌素D(ActD)诱导的凋亡作用。结论HBx可提高肝癌细胞的增殖活性,并增强肝癌细胞的抗凋亡能力,增加了肝癌细胞的恶性表型。

Objective Toexplore effects of hepatitis Bvirus Xprotein（HBx）on the malignantintensity of livercancercells.Methods The eukaryotic expression vector of pcDNAjHBx was constructed and transfected into HepG2 liver cancer cells by lipofetaminemediated method. The selected medium containing G418 was used to select the cell clones which express the X protein of HBV constantly. The HepG2 transfected with pcDNA3 was used as control. The integration of the exogenous vector DNA was detected by Neo gene polymerase chain reaction （PCR）.The expression of HBx protein was observed by fluorescence microscopy. Biological activity of pcDNAjHBx transfected HepG2 cells was evaluated by examination of cell proliferation, clone formation, MTT assay, Hoechst33342 staining,cell cycle and cell apoptosis. Results The MTT assay showed that the population growth rates of HepG2-HBx cells was faster than HepG2-pcDNA3 cells （28 h vs 32.5 h or 34 h ,P 〈 0.05 ）.The doubling time of HepG2- HBx cell was significantly shorter than that of HepG2-pcDNA3 cells. The clonogenicity of HepG2-HBx cells were increased [ （ 10.12 ＋ 0.23 ） % vs （5.33 ＋ O. 19） % or （5.19 ＋ 0.28） %, P 〈 0.05]. Flow cytometer （FCM） analysis showed that progression of HepG2-HBx cells from G0/G1 to S phase was promoted. The HepG2-HBx cells could resist the apoptosis induced by ActD. Conclusion The HBx protein could facilitate the proliferation of hepatocellular carcinoma cells and improve the ability of resisting apoptosis and enhance its malignant phenotype.