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
发布时间：2018-10-05 10:13
【摘要】：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(P0.01)。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)%,P0.01]。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)%,P0.05]。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(P0.01),24 h表达量最低(P0.01);P62蛋白在24 h出现最高表达水平(P0.01);而mTOR随着作用时间延长逐渐降低(48 h时,P0.01)。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P0.01],24 h相对减少[(12.000±2.828)vs(17.000±1.414),P0.01]。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。
[Abstract]:Aim: to investigate the effect of adenovirus ATV infection with apoptin gene (apopptin) on autophagy and apoptosis of human cervical cancer HeLa cells. Methods: HeLa cells were infected with adenovirus ATV carrying apoptin gene and control virus Ad-MOCK, respectively. The effect of ATV infection on the proliferation of HeLa cells was detected by WST-1 assay. The effect of ATV infection on apoptosis and cell cycle of HeLa cells was detected by flow cytometry. The effect of ATV infection on the expression of LC3,P62 and mTOR in HeLa cells was detected by Western blotting. The effect of ATV infection on the autophagy level of HeLa cells was detected by MDC staining. Results the proliferation of HeLa cells was inhibited by the infection of 1: ATV and had a time-effect relationship. The inhibitory rate of ATV 100 MOI infection for 72 h was 59.26, which was significantly higher than that in the control group (P0.01). The apoptosis rate of HeLa cells in the 48 h ATV group was significantly higher than that in the control group [(38.995 卤4.009) vs (14.680 卤1.174) P0.01]. With the prolongation of ATV, HeLa cells had the strongest arrest at 48 h in S phase, which was significantly higher than that in control group [(58.490 卤2.447) vs (43.235 卤4.419) P 0.05]. Compared with the control group, the expression level of LC3 increased gradually from 6h to 12h after ATV infection and reached the lowest (P0.01) at 24 h (P0.01) and the highest expression level of P62 protein (P0.01) at 24 h (P0.01), while mTOR decreased gradually (P0.01 at 48h). The positive staining of MDC in the perinuclear region of HeLa was observed under fluorescence microscope, and the number of autophagy bodies increased significantly with the prolongation of ATV action time, and the number of autophagy bodies increased significantly at 612h compared with the control group [(28.000 卤2.828) vs (8.500 卤2.121), (37.000 卤4.243) vs (14.000 卤1.414); P0.01] at 24 h, the number of autophagy bodies decreased relatively [(12.000 卤2.828) vs (17.000 卤1.414) P0.01]. Conclusion: ATV adenovirus carrying apoptin gene can promote apoptosis and autophagy of human cervical cancer HeLa cells and then specifically kill tumor cells.
【作者单位】： 长春中医药大学药学院;军事科学院军事兽医研究所分子病毒学与免疫学实验室;吉林医药学院检验学院;吉林省肿瘤医院头颈外一科;长春中医药大学基础医学院;
【基金】：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)~~
【分类号】：R737.33
[Abstract]:Aim: to investigate the effect of adenovirus ATV infection with apoptin gene (apopptin) on autophagy and apoptosis of human cervical cancer HeLa cells. Methods: HeLa cells were infected with adenovirus ATV carrying apoptin gene and control virus Ad-MOCK, respectively. The effect of ATV infection on the proliferation of HeLa cells was detected by WST-1 assay. The effect of ATV infection on apoptosis and cell cycle of HeLa cells was detected by flow cytometry. The effect of ATV infection on the expression of LC3,P62 and mTOR in HeLa cells was detected by Western blotting. The effect of ATV infection on the autophagy level of HeLa cells was detected by MDC staining. Results the proliferation of HeLa cells was inhibited by the infection of 1: ATV and had a time-effect relationship. The inhibitory rate of ATV 100 MOI infection for 72 h was 59.26, which was significantly higher than that in the control group (P0.01). The apoptosis rate of HeLa cells in the 48 h ATV group was significantly higher than that in the control group [(38.995 卤4.009) vs (14.680 卤1.174) P0.01]. With the prolongation of ATV, HeLa cells had the strongest arrest at 48 h in S phase, which was significantly higher than that in control group [(58.490 卤2.447) vs (43.235 卤4.419) P 0.05]. Compared with the control group, the expression level of LC3 increased gradually from 6h to 12h after ATV infection and reached the lowest (P0.01) at 24 h (P0.01) and the highest expression level of P62 protein (P0.01) at 24 h (P0.01), while mTOR decreased gradually (P0.01 at 48h). The positive staining of MDC in the perinuclear region of HeLa was observed under fluorescence microscope, and the number of autophagy bodies increased significantly with the prolongation of ATV action time, and the number of autophagy bodies increased significantly at 612h compared with the control group [(28.000 卤2.828) vs (8.500 卤2.121), (37.000 卤4.243) vs (14.000 卤1.414); P0.01] at 24 h, the number of autophagy bodies decreased relatively [(12.000 卤2.828) vs (17.000 卤1.414) P0.01]. Conclusion: ATV adenovirus carrying apoptin gene can promote apoptosis and autophagy of human cervical cancer HeLa cells and then specifically kill tumor cells.
【作者单位】： 长春中医药大学药学院;军事科学院军事兽医研究所分子病毒学与免疫学实验室;吉林医药学院检验学院;吉林省肿瘤医院头颈外一科;长春中医药大学基础医学院;
【基金】：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)~~
【分类号】：R737.33
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相关期刊论文 前3条
1 郭海宁;张萍;韩继成;靖杰;曹亮;肖朋朋;解长占;李一权;马海彬;南福龙;崔卓栋;李霄;田明尧;鲁会军;金宁一;;转瓶培养与生物反应器微载体培养重组腺病毒的比较[J];中国病原生物学杂志;2016年01期
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