集落形成法和Western Blot法研究ELAC2的辐射增敏作用  

Study the Radiosensitive Effect of ELAC2 by Using Colony Formation Assay and Western Blot Assay

作　　者：宁萍[1] 焦旸[1] 胡旭东[1] 朱巍[1] 徐加英[1] 樊赛军[1] 

机构地区：[1]苏州大学医学部放射医学与公共卫生学院江苏省放射医学与防护重点实验室,江苏苏州215123

出　　处：《中国辐射卫生》2010年第3期269-271,共3页Chinese Journal of Radiological Health

基　　金：国家自然科学基金项目(30701001);苏州大学医学发展基金项目(EE126708)

摘　　要：目的研究ELAC2基因对乳腺癌细胞MCF-7辐射敏感性的影响。方法利用脂质体转染法将ELAC2基因转入MCF-7细胞,同时设立未转染MCF-7和转染空载体的MCF-7/pcDNA3作为对照。RT-PCR法检测ELAC2基因的mRNA表达;集落形成法检测ELAC2基因对肿瘤细胞辐射敏感性的影响;Western Blot法检测ELAC2、FEN-1、MGMT、MRE11及Ku-70基因的蛋白表达水平。结果 ELAC2基因的mRNA和蛋白水平在转染细胞中表达明显增高。细胞接受0、0.5、1、2、4、6Gy的X射线照射后,ELAC2基因高表达使MCF-7细胞的剂量-效应曲线左移,细胞辐射敏感性增强。细胞经5Gy的X射线照射,ELAC2基因高表达未使MGMT蛋白和MRE11蛋白表达发生明显变化,而FEN-1蛋白及Ku-70蛋白表达水平显著下降。结论 ELAC2基因高表达使细胞辐射敏感性增高,其涉及的分子机制可能与ELAC2下调FEN-1蛋白及Ku-70蛋白表达水平有关。Objective To study the effects of ELAC2 gene on the human breast cancer cell MCF-7＇s radiosensitivity.Methods ELAC2 gene was transfected into MCF-7 via Lipofectamine transfection assay,while MCF-7 cells transfected with/without empty vector MCF-7/pcDNA3 were also established as negative control.The mRNA expression of ELAC2 gene was detected by RT-PCR assay.The colony formation assay was performed to test the radiosensitivity.The protein expression of ELAC2,FEN-1,MGMT,MRE11and Ku-70 were detected by western blot assay.Results Overexpressed ELAC2 level was both observed at mRNA and protein level of the cells transfected with pcDNA3.0/ELAC2.After 0,0.5,1,2,4,6Gy X-ray exposure,the radiation dose-response curve shifted to the left when ELAC2 overexpressed,and ELAC2 enhanced the radiosensitivity of MCF-7 cells.After 5Gy X-ray irradiation,no significant difference of MGMT and MRE11 protein expression levels were detected between ELAC2 overexpressed MCF-7 cells and control cells.While FEN-1 protein and Ku-70 protein expression levels were reduced significantly in MCF-7 cells with high level ELAC2.Conclusion ELAC2 gene could radiosensitize MCF-7 cells to X-ray irradiation,probably by the mechanisms of reducing the FEN-1 protein and Ku-70 protein expression.

关 键 词：ELAC2 乳腺癌 辐射敏感性 FEN-1 Ku-70 MGMT MRE11 

分 类 号：Q691.5[生物学—生物物理学]