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目的:构建表达重组反义p73基因的重组逆转录病毒,观察其对人肝癌细胞HepG2的凋亡诱导活性,并进一步探讨其作用机制。方法:克隆p73基因的反义片段,重组法构成逆转录病毒载体pBabe-p73(pBP73),以脂质体Lipofectamine2000将其转染293A细胞进行病毒包装;将逆转录病毒感染人肝癌细胞HepG2,用MTT法检测细胞生长抑制情况,Western blotting检测p73的表达;再分别用琼脂糖凝胶电泳和流式细胞仪检测肿瘤细胞的凋亡;最后检测p53,caspase-3和bcl-2蛋白的表达变化。结果重组质粒pBP73经鉴定连接正确,其转染293A细胞后上清液中可得到病毒,滴度达5×107pfu;MTT检测见pBP73病毒组48和72h细胞抑制率高于对照组(45.1%vs.5.3%,69.5%vs.17.3%,均p<0.05)。琼脂糖凝胶电泳出现典型梯形条带;流式细胞仪检测出现凋亡峰,于转染48h后达最高峰,其凋亡百分率高达20.47%;p73蛋白高表达组p53和caspase-3蛋白的表达亦有显著升高(p<0.05),但bcl-2蛋白未见表达差异。结论:成功构建了p73逆转录病毒,反义p73基因在体外能够有效地诱导人肝癌细胞HepG2的凋亡,其可能机制是通过激活caspase-3而发生作用。
OBJECTIVE: To construct a recombinant retrovirus expressing the recombinant antisense p73 gene and observe the apoptosis-inducing activity of HepG2 on human hepatocellular carcinoma cell line HepG2 and to explore its mechanism. Methods: The antisense fragment of p73 gene was cloned. The retroviral vector pBabe-p73 (pBP73) was constructed by recombination and transfected into 293A cells by Lipofectamine 2000. The retrovirus was used to infect HepG2 cells, MTT assay was used to detect the cell growth inhibition. Western blotting was used to detect the expression of p73. The apoptosis of tumor cells was detected by agarose gel electrophoresis and flow cytometry respectively. The expressions of p53, caspase-3 and bcl-2 were detected . Results The recombinant plasmid pBP73 was identified and ligated correctly. The virus in the supernatant was transfected into 293A cells with a titer of 5 × 107 pfu. The inhibitory rates of pBP73 at 48 and 72 h were higher than that of the control (45.1% vs .5.3%, 69.5% vs.17.3%, all p <0.05). A typical trapezoidal band appeared on agarose gel electrophoresis. The peak of apoptosis was detected by flow cytometry, which peaked at 48h after transfection. The percentage of apoptotic cells was up to 20.47%. The expression of p53 and caspase-3 protein in high expression of p73 protein The expression of bcl-2 protein was also significantly increased (p <0.05), but there was no difference in bcl-2 protein. CONCLUSION: The p73 retrovirus was successfully constructed and the antisense p73 gene could effectively induce the apoptosis of HepG2 cells in vitro. The possible mechanism is that caspase-3 activates caspase-3.