共转染CDK1、CDK2siRNA对肿瘤细胞周期和细胞凋亡的影响  

Influence of Cotransfected CDK1 and CDK2 siRNA on Tumor Cell Cycle and Apoptosis

作　　者：肖徽[1] 魏欣[1] 于东东[1] 徐向上[1] 黄丹[1] 王贵华[1] 李小兰[1] 陶德定[1] 胡俊波[1] 龚建平[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院分子医学中心,肿瘤研究所,武汉市430030

出　　处：《医学分子生物学杂志》2009年第6期473-478,共6页Journal of Medical Molecular Biology

基　　金：资助项目：国家重点基础研究发展规划（973计划）（No.2004CB518705,Nn2002CB513100）,国家自然科学基金（No.30570908）

摘　　要：目的研究共转染CDK1、CDK2siRNA同时抑制CDKI、CDK2蛋白表达对肿瘤细胞周期和细胞凋亡的影响，探讨细胞周期主要调控分子在肿瘤细胞凋亡中的作用。方法以人宫颈癌细胞株HeLa细胞为研究对象，用脂质体lipofectamine2000同时转染CDKl和CDK2siRNA。在转染后48、60h收集细胞，用Western印迹检测CDKl、CDK2蛋白的表达，AnnexinV／PI检测转染细胞的凋亡，流式细胞术DNA含量检测分析细胞周期。转染细胞进行瑞氏一姬姆萨染色（Wright—Giemsa）后在显微镜下观察其形态变化i结果共转染CDKl、CDK2siRNA后48和60h，Western印迹结果显示CDKl和CDK2蛋白的表达都同时降低。共转染CDKl、CDK2siRNA后，细胞周期S期和G1／M期比例与对照相比有明显增加；共转染细胞经瑞氏一姬姆萨染色后在显微镜下可见双核或多核细胞增多；AnnexinV／PI检测结果显示共转染CDK1、CDK2siRNA的细胞在48和60h细胞凋亡率与对照相比有显著的升高。结论siRNA干扰导致的CDKI、CDK2表达同时降低不仅导致细胞周期s期和G1／M期的阻滞，也诱导了肿瘤细胞的凋亡。Objective To investigate the influence of CDK1 and CDK2 siRNA knockdown on cell cycle and apoptosis, and explore the role of cell cycle master regulators in tumor cell apopto- sis. Methods The siRNA% targeting CDK1 and CDK2 genes were synthesized and simultaneously cotransfected into Hela cells by lipofectamine2000. 48 and 60 hours after cotransfection, expres- sions of CDK1 and CDK2 were examined by Western blot. Cell cycle arrest was analyzed by flow ey- tometry. Apoptosis was detected by Annexin V/PI method. Morphological changes of transfected cells were stained with Wright-Giemsa Staining and studied under a microscope. Results CDK1 and CDK2 protein expression was down-regulated at 48 and 60 hours after siRNA cotransfection. G2/M and S phase cell cycle population in the cotransfected cells was obviously enhanced compared with control. Annexirr/IP analysis revealed that apoptotie induction of the cotransfected cells was signifi- cantly increased compared with control. More binueleate and muhinucleate cells were observed in the cotransfeeted cells under the microscope. Conclusion The expression of CDK1 and CDK2 was decreased by cotransfection of CDK1 and CDK2 siRNA, leading to not only tumor cell cycle arrest in S phase and G2/M phase, but also tumor cell apoptosis.

关 键 词：细胞周期依赖性蛋白激酶 细胞周期阻滞 肿瘤细胞凋亡 RNA干扰 

分 类 号：R349.1[医药卫生—基础医学]