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.

国家重点研发计划(973 计划）资助项目（No. 2016YSC1200900）；国家“重大新药创制”科技重大专项资助（No．2014ZX09304314）；吉林省重大科技攻关项目资助（No. 20150201002YY）；吉林省产业技术创新战略联盟项目资助（No. 20140309006YY）；长春市重大科技攻关项目资助（No.16ss11）；吉林省科技发展计划项目资助（No. 20150101124JC）