Establishment of Hepatocellular Carcinoma Cell Line HepG2 Stably Transfected by Hepatitis B Virus X Gene and Its Effects on p53-p21 Pathway
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摘要: 目的 将构建的乙型肝炎病毒X基因的真核表达载体稳定转染HepG2细胞株, 并研究转染后对p53-p21途径的影响。 方法 用基因转染的方法将已构建的载体pcDNA3.1-HBx 转染入肝癌细胞HepG2 中, G418筛选出稳定转染X基因的细胞株。细胞生长曲线、平板克隆形成实验检测转染后肝癌细胞的恶性表型，流式细胞仪检测细胞凋亡和周期，Western blot检测p53蛋白的表达，RT-PCR检测p21基因。结果 成功筛选出pcDNA3.1-HBx稳定转染的HepG2细胞株；细胞生长实验、平板克隆形成实验显示稳定转染乙型肝炎病毒X基因的肝癌细胞恶性表型增高；流式细胞仪结果显示，转染后的细胞株凋亡率增高，G1/G0期细胞减少，G2期细胞增多；Western blot 和RT-PCR分别显示，转染株p53蛋白表达增高，且下调p21基因水平。 结论 乙型肝炎病毒X蛋白可刺激肝癌细胞生长, 稳定转染X基因的细胞株可能通过p53-p21途径增加肝癌细胞的恶性表型。Abstract: Objective To establish the HepG cell line with stably transfected hepatitis B virus X gene and to study its effects on the p53-p21 pathway. Methods Lipofectamine was used to transfect the eukaryotic expression vector pcDNA3.1-HBx into human hepatocellular carcinoma cell line HepG2. G418 was used to select the cell clones for stable expression of the HBx protein of HBV. Cell growth curve and clone plate experiment were used to analyse the malignant phenotype and the growth of hepatocellular carcinoma cells. Flow cytometry was used to assay the apoptosis rate and cell cycle. p53 protein expression levels were detected by Western blot and p21mRNA levels were detected by RT-PCR. Results pcDNA3.1-HBx gene HepG2 cell line was successfully established. The cell growth and clone plate assay revealed that these cells had a relatively increased malignant phenotype. The results of fl ow cytometry also manifested that apoptosis rate of these transfected cells were higher than that of HepG2 cells. The cell number of G1/G0 cycle phase decreased and the cell number of G2 cycle phase increased, while the expression of p53 protein increased and p21 mRNA decreased in those cells. Conclusion The HBx protein could promote the growth of hepatocellular carcinoma cells. The cell line stably tansfected by HBx gene could enhance its malignancy through p53-p21 pathway.
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Key words:
- Hepatitis B virus x gene /
- Eukaryotic expression vector /
- HepG2 cell /
- p53-p21 pathway
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