反义p73基因诱导人肝癌细胞HepG2的凋亡

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。

Apoptosis-inducing Effect of Anti-p73 Gene on Human Colon Cancer Cell HepG2

Objective: To discuss the mechanism of p73 inducing tumor cells into apoptosis by constructing recombinant expression plasmid pBP73 and observing the expression and apoptosis-inducing effect of anti-p73 gene on colon cancer cell HepG2 Methods: Anti-p73 gene was cloned into the plasmid pBabe to form the recombinant plasmid pBP73,and then pBP73 was transformed into HepG2 cells and then agarose electrophoresis and flow cytometry were used to verify apoptosis of tumor cells.At last caspase-3 was detected by Wes...