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1356~1361.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].尹逊哲,陈爽,李文杰,朱羿龙,李一权,崔传信,李敏,崔英丽,赵津,李善智,郑颖,孙丽丽,李霄,郭焱,金宁一.中国肿瘤生物治疗杂志,2017,24(12)
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    点此下载全文    点此浏览HTML全文
尹逊哲  陈爽  李文杰  朱羿龙  李一权  崔传信  李敏  崔英丽  赵津  李善智  郑颖  孙丽丽  李霄  郭焱  金宁一
1.长春中医药大学药学院,吉林长春 130117;2.军事科学院军事兽医研究所分子病毒学与免疫学实验室,吉林长春130122;3.长春中医药大学基础医学院,吉林长春130117;4.吉林医药学院检验学院,吉林吉林132013;5. 吉林省肿瘤医院头颈外一科,吉林长春130021,1.长春中医药大学药学院,吉林长春 130117;2.军事科学院军事兽医研究所分子病毒学与免疫学实验室,吉林长春130122;3.长春中医药大学基础医学院,吉林长春130117;4.吉林医药学院检验学院,吉林吉林132013;5. 吉林省肿瘤医院头颈外一科,吉林长春130021,1.长春中医药大学药学院,吉林长春 130117;2.军事科学院军事兽医研究所分子病毒学与免疫学实验室,吉林长春130122;3.长春中医药大学基础医学院,吉林长春130117;4.吉林医药学院检验学院,吉林吉林132013;5. 吉林省肿瘤医院头颈外一科,吉林长春130021,1.长春中医药大学药学院,吉林长春 130117;2.军事科学院军事兽医研究所分子病毒学与免疫学实验室,吉林长春130122;3.长春中医药大学基础医学院,吉林长春130117;4.吉林医药学院检验学院,吉林吉林132013;5. 吉林省肿瘤医院头颈外一科,吉林长春130021,1.长春中医药大学药学院,吉林长春 130117;2.军事科学院军事兽医研究所分子病毒学与免疫学实验室,吉林长春130122;3.长春中医药大学基础医学院,吉林长春130117;4.吉林医药学院检验学院,吉林吉林132013;5. 吉林省肿瘤医院头颈外一科,吉林长春130021,1.长春中医药大学药学院,吉林长春 130117;2.军事科学院军事兽医研究所分子病毒学与免疫学实验室,吉林长春130122;3.长春中医药大学基础医学院,吉林长春130117;4.吉林医药学院检验学院,吉林吉林132013;5. 吉林省肿瘤医院头颈外一科,吉林长春130021,1.长春中医药大学药学院,吉林长春 130117;2.军事科学院军事兽医研究所分子病毒学与免疫学实验室,吉林长春130122;3.长春中医药大学基础医学院,吉林长春130117;4.吉林医药学院检验学院,吉林吉林132013;5. 吉林省肿瘤医院头颈外一科,吉林长春130021,1.长春中医药大学药学院,吉林长春 130117;2.军事科学院军事兽医研究所分子病毒学与免疫学实验室,吉林长春130122;3.长春中医药大学基础医学院,吉林长春130117;4.吉林医药学院检验学院,吉林吉林132013;5. 吉林省肿瘤医院头颈外一科,吉林长春130021,1.长春中医药大学药学院,吉林长春 130117;2.军事科学院军事兽医研究所分子病毒学与免疫学实验室,吉林长春130122;3.长春中医药大学基础医学院,吉林长春130117;4.吉林医药学院检验学院,吉林吉林132013;5. 吉林省肿瘤医院头颈外一科,吉林长春130021,1.长春中医药大学药学院,吉林长春 130117;2.军事科学院军事兽医研究所分子病毒学与免疫学实验室,吉林长春130122;3.长春中医药大学基础医学院,吉林长春130117;4.吉林医药学院检验学院,吉林吉林132013;5. 吉林省肿瘤医院头颈外一科,吉林长春130021,1.长春中医药大学药学院,吉林长春 130117;2.军事科学院军事兽医研究所分子病毒学与免疫学实验室,吉林长春130122;3.长春中医药大学基础医学院,吉林长春130117;4.吉林医药学院检验学院,吉林吉林132013;5. 吉林省肿瘤医院头颈外一科,吉林长春130021,1.长春中医药大学药学院,吉林长春 130117;2.军事科学院军事兽医研究所分子病毒学与免疫学实验室,吉林长春130122;3.长春中医药大学基础医学院,吉林长春130117;4.吉林医药学院检验学院,吉林吉林132013;5. 吉林省肿瘤医院头颈外一科,吉林长春130021,1.长春中医药大学药学院,吉林长春 130117;2.军事科学院军事兽医研究所分子病毒学与免疫学实验室,吉林长春130122;3.长春中医药大学基础医学院,吉林长春130117;4.吉林医药学院检验学院,吉林吉林132013;5. 吉林省肿瘤医院头颈外一科,吉林长春130021,1.长春中医药大学药学院,吉林长春 130117;2.军事科学院军事兽医研究所分子病毒学与免疫学实验室,吉林长春130122;3.长春中医药大学基础医学院,吉林长春130117;4.吉林医药学院检验学院,吉林吉林132013;5. 吉林省肿瘤医院头颈外一科,吉林长春130021,1.长春中医药大学药学院,吉林长春 130117;2.军事科学院军事兽医研究所分子病毒学与免疫学实验室,吉林长春130122;3.长春中医药大学基础医学院,吉林长春130117;4.吉林医药学院检验学院,吉林吉林132013;5. 吉林省肿瘤医院头颈外一科,吉林长春130021
基金项目: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)
DOI:10.3872/j.issn.1007-385X.2017.12.003
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(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。
关键词:宫颈癌  HeLa细胞  凋亡素基因  溶瘤腺病毒  自噬  凋亡
Inhibitory effect of apopotin-loaded oncolytic adenovirus ATV on human cervical carcinoma HeLa cells    Download Fulltext
YIN Xunzhe  CHEN Shuang  LI Wenjie  ZHU Yilong  LI Yiquan  CUI Chuanxin  LI Min  CUI Yingli  ZHAO Jin  LI Shanzhi  ZHENG Ying  SUN Lili  LI Xiao  GUO Yan  JIN Ningyi
1.College of Pharmacy,Changchun University of Traditional Chinese Medicine, Changchun 130117, Jilin, China; 2. Molecular Virology and Immunology Laboratory, Institute of Military Veterinary and Academy of Military Medical Sciences, Changchun 130122, Jilin, China; 3. College of Basic Medicine, Changchun University of Traditional Chinese Medicine,Changchun 130117, Jilin, China; 4. College of Examination, Jilin Medical College, Jilin 132013, Jilin, China;5. Department of Head and Neck Surgery, Jilin Tumor Hospital, Changchun 130021, Jilin, China,1.College of Pharmacy,Changchun University of Traditional Chinese Medicine, Changchun 130117, Jilin, China; 2. Molecular Virology and Immunology Laboratory, Institute of Military Veterinary and Academy of Military Medical Sciences, Changchun 130122, Jilin, China; 3. College of Basic Medicine, Changchun University of Traditional Chinese Medicine,Changchun 130117, Jilin, China; 4. College of Examination, Jilin Medical College, Jilin 132013, Jilin, China;5. Department of Head and Neck Surgery, Jilin Tumor Hospital, Changchun 130021, Jilin, China,1.College of Pharmacy,Changchun University of Traditional Chinese Medicine, Changchun 130117, Jilin, China; 2. Molecular Virology and Immunology Laboratory, Institute of Military Veterinary and Academy of Military Medical Sciences, Changchun 130122, Jilin, China; 3. College of Basic Medicine, Changchun University of Traditional Chinese Medicine,Changchun 130117, Jilin, China; 4. College of Examination, Jilin Medical College, Jilin 132013, Jilin, China;5. Department of Head and Neck Surgery, Jilin Tumor Hospital, Changchun 130021, Jilin, China,1.College of Pharmacy,Changchun University of Traditional Chinese Medicine, Changchun 130117, Jilin, China; 2. Molecular Virology and Immunology Laboratory, Institute of Military Veterinary and Academy of Military Medical Sciences, Changchun 130122, Jilin, China; 3. College of Basic Medicine, Changchun University of Traditional Chinese Medicine,Changchun 130117, Jilin, China; 4. College of Examination, Jilin Medical College, Jilin 132013, Jilin, China;5. Department of Head and Neck Surgery, Jilin Tumor Hospital, Changchun 130021, Jilin, China,1.College of Pharmacy,Changchun University of Traditional Chinese Medicine, Changchun 130117, Jilin, China; 2. Molecular Virology and Immunology Laboratory, Institute of Military Veterinary and Academy of Military Medical Sciences, Changchun 130122, Jilin, China; 3. College of Basic Medicine, Changchun University of Traditional Chinese Medicine,Changchun 130117, Jilin, China; 4. College of Examination, Jilin Medical College, Jilin 132013, Jilin, China;5. Department of Head and Neck Surgery, Jilin Tumor Hospital, Changchun 130021, Jilin, China,1.College of Pharmacy,Changchun University of Traditional Chinese Medicine, Changchun 130117, Jilin, China; 2. Molecular Virology and Immunology Laboratory, Institute of Military Veterinary and Academy of Military Medical Sciences, Changchun 130122, Jilin, China; 3. College of Basic Medicine, Changchun University of Traditional Chinese Medicine,Changchun 130117, Jilin, China; 4. College of Examination, Jilin Medical College, Jilin 132013, Jilin, China;5. Department of Head and Neck Surgery, Jilin Tumor Hospital, Changchun 130021, Jilin, China,1.College of Pharmacy,Changchun University of Traditional Chinese Medicine, Changchun 130117, Jilin, China; 2. Molecular Virology and Immunology Laboratory, Institute of Military Veterinary and Academy of Military Medical Sciences, Changchun 130122, Jilin, China; 3. College of Basic Medicine, Changchun University of Traditional Chinese Medicine,Changchun 130117, Jilin, China; 4. College of Examination, Jilin Medical College, Jilin 132013, Jilin, China;5. Department of Head and Neck Surgery, Jilin Tumor Hospital, Changchun 130021, Jilin, China,1.College of Pharmacy,Changchun University of Traditional Chinese Medicine, Changchun 130117, Jilin, China; 2. Molecular Virology and Immunology Laboratory, Institute of Military Veterinary and Academy of Military Medical Sciences, Changchun 130122, Jilin, China; 3. College of Basic Medicine, Changchun University of Traditional Chinese Medicine,Changchun 130117, Jilin, China; 4. College of Examination, Jilin Medical College, Jilin 132013, Jilin, China;5. Department of Head and Neck Surgery, Jilin Tumor Hospital, Changchun 130021, Jilin, China,1.College of Pharmacy,Changchun University of Traditional Chinese Medicine, Changchun 130117, Jilin, China; 2. Molecular Virology and Immunology Laboratory, Institute of Military Veterinary and Academy of Military Medical Sciences, Changchun 130122, Jilin, China; 3. College of Basic Medicine, Changchun University of Traditional Chinese Medicine,Changchun 130117, Jilin, China; 4. College of Examination, Jilin Medical College, Jilin 132013, Jilin, China;5. Department of Head and Neck Surgery, Jilin Tumor Hospital, Changchun 130021, Jilin, China,1.College of Pharmacy,Changchun University of Traditional Chinese Medicine, Changchun 130117, Jilin, China; 2. Molecular Virology and Immunology Laboratory, Institute of Military Veterinary and Academy of Military Medical Sciences, Changchun 130122, Jilin, China; 3. College of Basic Medicine, Changchun University of Traditional Chinese Medicine,Changchun 130117, Jilin, China; 4. College of Examination, Jilin Medical College, Jilin 132013, Jilin, China;5. Department of Head and Neck Surgery, Jilin Tumor Hospital, Changchun 130021, Jilin, China,1.College of Pharmacy,Changchun University of Traditional Chinese Medicine, Changchun 130117, Jilin, China; 2. Molecular Virology and Immunology Laboratory, Institute of Military Veterinary and Academy of Military Medical Sciences, Changchun 130122, Jilin, China; 3. College of Basic Medicine, Changchun University of Traditional Chinese Medicine,Changchun 130117, Jilin, China; 4. College of Examination, Jilin Medical College, Jilin 132013, Jilin, China;5. Department of Head and Neck Surgery, Jilin Tumor Hospital, Changchun 130021, Jilin, China,1.College of Pharmacy,Changchun University of Traditional Chinese Medicine, Changchun 130117, Jilin, China; 2. Molecular Virology and Immunology Laboratory, Institute of Military Veterinary and Academy of Military Medical Sciences, Changchun 130122, Jilin, China; 3. College of Basic Medicine, Changchun University of Traditional Chinese Medicine,Changchun 130117, Jilin, China; 4. College of Examination, Jilin Medical College, Jilin 132013, Jilin, China;5. Department of Head and Neck Surgery, Jilin Tumor Hospital, Changchun 130021, Jilin, China,1.College of Pharmacy,Changchun University of Traditional Chinese Medicine, Changchun 130117, Jilin, China; 2. Molecular Virology and Immunology Laboratory, Institute of Military Veterinary and Academy of Military Medical Sciences, Changchun 130122, Jilin, China; 3. College of Basic Medicine, Changchun University of Traditional Chinese Medicine,Changchun 130117, Jilin, China; 4. College of Examination, Jilin Medical College, Jilin 132013, Jilin, China;5. Department of Head and Neck Surgery, Jilin Tumor Hospital, Changchun 130021, Jilin, China,1.College of Pharmacy,Changchun University of Traditional Chinese Medicine, Changchun 130117, Jilin, China; 2. Molecular Virology and Immunology Laboratory, Institute of Military Veterinary and Academy of Military Medical Sciences, Changchun 130122, Jilin, China; 3. College of Basic Medicine, Changchun University of Traditional Chinese Medicine,Changchun 130117, Jilin, China; 4. College of Examination, Jilin Medical College, Jilin 132013, Jilin, China;5. Department of Head and Neck Surgery, Jilin Tumor Hospital, Changchun 130021, Jilin, China and 1.College of Pharmacy,Changchun University of Traditional Chinese Medicine, Changchun 130117, Jilin, China; 2. Molecular Virology and Immunology Laboratory, Institute of Military Veterinary and Academy of Military Medical Sciences, Changchun 130122, Jilin, China; 3. College of Basic Medicine, Changchun University of Traditional Chinese Medicine,Changchun 130117, Jilin, China; 4. College of Examination, Jilin Medical College, Jilin 132013, Jilin, China;5. Department of Head and Neck Surgery, Jilin Tumor Hospital, Changchun 130021, Jilin, China
Fund Project: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)
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 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.
Keywords:cervical cancer  HeLa cell  apoptin gene  oncolytic adenovirus  autophagy  apoptosis
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