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
摘要
Abstract
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 100MOI 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＜O.0 1].Conclusion:Apoptin-loaded oncolytic adenovirus ATV can promote the autophagy and apoptosis of human cervical cancer HeLa cells and then specifically kill tumor cells.关键词
宫颈癌/HeLa细胞/凋亡素基因/溶瘤腺病毒/自噬/凋亡Key words
cervical cancer/HeLa cell/apoptin gene/oncolytic adenovirus/autophagy/apoptosis分类
医药卫生引用本文复制引用
尹逊哲,李善智,郑颖,孙丽丽,李霄,郭焱,金宁一,陈爽,李文杰,朱羿龙,李一权,崔传信,李敏,崔英丽,赵津..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[J].中国肿瘤生物治疗杂志,2017,24(12):1356-1361,6.基金项目
国家重点研发计划(973计划)资助项目(No.2016YSC 1200900) （973计划）
国家“重大新药创制”科技重大专项资助(No.2014ZX09304314) （No.2014ZX09304314）
吉林省重大科技攻关项目资助(No.20150201002YY) （No.20150201002YY）
吉林省产业技术创新战略联盟项目资助(No.20140309006YY) （No.20140309006YY）
长春市重大科技攻关项目资助(No.16ss11) （No.16ss11）
吉林省科技发展计划项目资助(No.20150101124JC). Project supported by the National Key R&D Program (973 Program) of China (No.2016YSC1200900),the Major New Drugs Innovation and Development(No.2014ZX09304314),the Key Technologies R&D Program of Jilin Province (No.0150201002YY),the Industrial Technology Innovation Strategic Alliance Project of Jilin Province (No.20140309006YY),the Major Scientific and Technological Project of Changchun City(No.16ss11),and the Technology Development Project of Jilin Province(No.20150101124JC) （No.20150101124JC）
