重组人p66Shc腺病毒抑制HeLa细胞增殖的机制研究

Recombinant adenovirus of human p66Shc inhibits proliferation in HeLa cells

目的探讨重组人p66Shc腺病毒(AdenoⅩ-p66Shc)对宫颈癌HeLa细胞增殖的抑制作用及机制。方法MTT法检测细胞活力,DCFH-DA荧光探针检测活性氧(ROS)生成;ELISA检测8-羟基脱氧鸟苷(8-OHdG)含量;AnnexinⅤ-FITC/PI荧光染色检测细胞凋亡率;Western blot检测相关蛋白表达;免疫共沉淀检测p66Shc与p53之间相互作用。结果重组人p66Shc腺病毒呈剂量依赖性地抑制HeLa细胞增殖(P<0.05),而N-乙酰半胱氨酸能够阻断重组人p66Shc腺病毒的作用。p66Shc能引起细胞ROS水平显著上升(P<0.05),同时伴随8-OHdG含量的升高(P<0.05);p66Shc能引起p53蛋白和CyclinB1蛋白表达及p53磷酸化修饰(p-p53)显著升高(P<0.05),但免疫共沉淀实验结果显示p66Shc与p53并非直接结合,提示p66Shc通过其他信号分子间接调控p53的表达及活性。结论重组人p66Shc腺病毒通过使HeLa细胞ROS水平上升,引起DNA氧化损伤,从而诱导p53表达及其磷酸化修饰升高抑制HeLa细胞增殖。

Objective To determine the inhibitory effect of a recombinant adenovirus of human p66Shc （Adeno X-p66She ） on the proliferation of human eervieal eaneer HeLa cells and investigate the possible underlying mechanism. Methods The Hela cells were infected with Adeno X-LacZ and Adeno X-p66She respectively. The eells without infeetion served as blank control. Cell viability was analyzed by MTT assay. Reactive oxygen species （ROS） was measured by DCFH-DA fluorescent probes and flow cytometry. The levels of 8-OHdG were detected by ELISA assay. Cell apoptosis was detected by Annexin V-FITC/PI staining. Western blotting was used to observe the expression of associated proteins. Co-immunopreeipitation （Co-IP） was used to investigate the possible interaction between p66She and p53. Results MTT assay showed that the proliferation of HeLa cells was significantly suppressed （ P 〈 0.05 ） by Adeno X-p66She in a dose-dependent manner, and its inhibition eould be blocked by N-aeetyl-L-eysteine. Meanwhile, the levels of ROS and 8-OHdG were significantly increased after Adeno X-p66Shc infection （ P 〈 0.05 ）. The expression of p53, Cyelin B1 and p-p53 was up-regulated with the over-expression of p66She, but the result of Co-IP demonstrated that p66Shc and p53 were not direetly eombined, suggesting the expression and activity of p53 was regulated by other molecular aetivated by p66She. Conclusion Adeno X-p66She induces ROS generation and DNA oxidative damage, and up-regulates the expression of p53 and p-p53, and thereby inhibits the proliferation of HeLa eells.