文章摘要
马丽思,翁怀玉,刘思旋,陈 璇,宗祥云.SETD8基因表达对乳腺癌细胞周期、活性氧及药物敏感性的影响[J].,2020,(12):2201-2206
SETD8基因表达对乳腺癌细胞周期、活性氧及药物敏感性的影响
Effect of SETD8 Gene Expression on Cell Cycle, Reactive Oxygen Species and Drug Sensitivity of Breast Cancer Cells
投稿时间:2020-02-25  修订日期:2020-03-21
DOI:10.13241/j.cnki.pmb.2020.12.001
中文关键词: 慢病毒  细胞周期  SETD8  敏感性  活性氧
英文关键词: Lentivirus  Cell cycle  SETD8  Drug sensitivity  ROS
基金项目:国家自然科学基金项目(8177101282)
作者单位E-mail
马丽思 上海交通大学附属第六人民医院普外科 上海 200233 965091200@qq.com 
翁怀玉 上海交通大学附属第六人民医院普外科 上海 200233  
刘思旋 上海交通大学附属第六人民医院普外科 上海 200233  
陈 璇 上海交通大学附属第六人民医院普外科 上海 200233  
宗祥云 上海交通大学附属第六人民医院普外科 上海 200233  
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中文摘要:
      摘要 目的:构建SETD8重组慢病毒载体,探讨SETD8对人乳腺癌细胞周期、氧化应激的影响,并观察稳定敲低SETD8后对乳腺癌细胞多西他赛敏感性的影响。方法:构建含SETD8基因的重组慢病毒载体,经转染,荧光显微镜观察感染效率;采用RT-qPCR和Western blot 检测SETD8 mRNA和蛋白相对表达量;采用流式细胞技术检测细胞周期及活性氧水平,通过CCK-8试剂检测稳定敲低SETD8基因的乳腺癌细胞对多西他赛的敏感性变化。结果:成功包装慢病毒,荧光观察慢病毒感染效率在85%左右,通过PCR和WB验证 SETD8过表达及敲低稳转细胞系的效率显著(P<0.01)。低表达SETD8的乳腺癌细胞周期停滞在G2/M和S期,细胞内ROS水平高于对照组。不同浓度多西他赛处理的SETD8敲低稳转细胞系的细胞活性较对照组明显降低(P<0.01)。结论:慢病毒介导下调SETD8表达,使得乳腺癌细胞周期阻滞在G2/M期,细胞内ROS增多,与多西他赛发生协同作用,从而增加了药物敏感性。
英文摘要:
      ABSTRACT Objective: To construct a recombinant lentivirus vector of SETD8, to investigate the effect of SETD8 on the cell cycle and oxidative stress of human breast cancer, and to observe the effect of stable SETD8 knockdown on docetaxel sensitivity of breast carcinoma cells. Methods: Recombinant lentivirus vector containing SETD8 gene was constructed, transfected into cancer cells and the infection efficiency was observed by fluorescence microscopy. The relative mRNA and protein expressions of SETD8 were detected by RT-qPCR and Western blot. Flow cytometry cell cycle and reactive oxygen species levels were evaluated by flow cytometry. Changes in docetaxel sensitivity of breast cancer cells with stable SETD8 knockdown were detected by the CCK8 kit. Results: Lentivirus were successfully packaged, the infection efficiency of lentivirus was about 85% by fluorescence observation. The relative expression of SETD8 in MDA-MB-231-shSETD8 and MCF-7-SETD8 cells was significantly lower or higher than in control cells(P<0.01). Compared with control cells, the biological behavior of MDA-MB-231-shSETD8 reduced, the cells at S and G2/M phase increased, and intracellular ROS level was also increased. The cell viability of MDA-MB-231-shSETD8 treated with docetaxel at different concentrations was significantly lower than that of the control group (P<0.01). Conclusion: SETD8 recombinant lentiviral vector transfected breast carcinoma cells, successfully established the stable knockdown cell lines, it reduce the malignant biological behavior and enhance the in vitro inhibitory effects of docetaxel on breast carcinoma cell lines.
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