携带凋亡素基因的溶瘤腺病毒ATV对人宫颈癌HeLa细胞的抑制作用

Inhibitory effect of apopotin-loaded oncolytic adenovirus ATV on human cervical carcinoma HeLa cells

目的:探讨携带凋亡素基因(apopptin)的溶瘤腺病毒ATV感染对人宫颈癌HeLa细胞自噬和凋亡的影响。方法:分别用携带凋亡素基因的溶瘤腺病毒ATV与对照病毒Ad-MOCK(均为前期工作构建)感染HeLa细胞后,应用WST-1法检测ATV感染对HeLa细胞增殖的影响,流式细胞术检测ATV感染对HeLa细胞凋亡和细胞周期的影响,Western blotting法检测ATV感染对HeLa细胞自噬相关蛋白LC3、P62与mTOR表达的影响,MDC染色法检测ATV感染对HeLa细胞自噬水平的影响。结果:ATV感染后抑制HeLa细胞增殖,并具有一定的时间效应关系;以ATV 100 MOI感染72 h后抑制率达到59.26%,其抑制能力明显强于同时间段对照组(P<0.01)。ATV感染48 h,ATV组HeLa细胞凋亡率显著高于对照组[(38.995±4.009)%vs(14.680±1.174)%,P<0.01]。感染后的HeLa细胞,随着ATV作用时间的延长,S期出现阻滞,48 h阻滞最强,显著高于对照组[(58.490±2.447)%vs(43.235±4.419)%,P<0.05]。与对照组相比,ATV感染HeLa后,LC3表达量6、12 h逐渐增高,12 h达到最大值(P<0.01),24 h表达量最低(P<0.01);P62蛋白在24 h出现最高表达水平(P<0.01);而mTOR随着作用时间延长逐渐降低(48 h时,P<0.01)。荧光显微镜下可见HeLa细胞核周区域MDC阳性染色,随着ATV作用时间延长,自噬小体明显增多;与对照组相比,6、12h自噬小体数量显著增多[(28.000±2.828)vs(8.500±2.121),(37.000±4.243)vs(14.000±1.414);均P<0.01],24 h相对减少[(12.000±2.828)vs(17.000±1.414),P<0.01]。结论:携带凋亡素基因的ATV溶瘤腺病毒可促进人宫颈癌HeLa细胞的凋亡和自噬水平,进而特异性杀伤肿瘤细胞。

Objective： To investigate the effect of apoptin-loaded oncolytic adenovirus ATV infection on the autophagy and apoptosis of human cervical carcinoma HeLa cells. Methods： Apoptin- loaded oncolytic adenovirus ATV and the control virus Ad-MOCK （both were constructed previously） were transfected into HeLa cells. The effect of ATV infection on proliferation of HeLa cells was measured through WST-1 assay; The effect of ATV infection on apoptosis and cell cycle of HeLa cells was detected by flow cytometry; Western blotting was used to detect the effect of ATV infection on the expression of autophagy-related proteins （LC3, P62 and mTOR） in HeLa cells;MDC staining was used to detect the effect of ATV infection on autophagy of HeLa cells. Results： ATV infection inhibited HeLa cell proliferation in a time-dependent manner; and the inhibitory rate reached 59.26% with ATV 100 MOI at 72 h, which was significantly higher than that of the control group （P〈0.01）; The apoptotic rate of HeLa cells in ATV group was significantly higher than that in control group[ （38.995±4.009）% vs（14.680±1.174）%,P〈0.01] 48 h after infection. With the infection of ATV, flow cytometry analysis showed that the cell cycle of HeLa was blocked at S phase, which was most significant at 48 h and significantly higher than that in control group [ （58.490±2.447）% vs （43.235±4.419）%, P〈0.05]. Western blotting revealed that after ATV infection, LC3 expression was gradually increase at 6, 12 h, with a highest level at 12 h（0.368±0.010,P〈0.01）and a lowest level at 24 h （0.106 ± 0.023,P〈0.01）,P62 protein showed the highest expression level at 24 h（6.004 ± 1.423,P〈0.01）, while mTOR gradually reduced（0.042±0.010,48 h,P〈0.01）with the extension of infection time; under fluorescent microscope,monodansylcadaverin （MDC） positive staining of autophagosomes was observed in the peripheral region of the nucleus of HeLa cells; the number of autophagosomes of ATV group was significantly increased as the ATV infection time prolonged; compared with control group, the number of autophagosomes of ATV group significantly increased at 6 h and 12 h [ （28.000±2.828） vs （8.500±2.121）, （37.000±4.243） vs （14.000±1.414）, P〈0.01], but relatively reduced at 24 h [ （12.000±2.828） vs （17.000±1.414）, P〈0.01]. Conclusion： Apoptin-loaded oncolytic adenovirus ATV can promote the autophagy and apoptosis of human cervical cancer HeLa cells and then specifically kill tumor cells.