慢病毒介导CDK1/CDK2干扰对肿瘤细胞周期的影响  

Effect of CDK1/CDK2 interference on cell cycle by lentivirus vector in cancer cells

作　　者：江文娇 李慧萍[1] 齐庆远[1] 

机构地区：[1]浙江师范大学化学与生命科学学院,浙江金华321004

出　　处：《浙江师范大学学报（自然科学版）》2015年第3期348-353,共6页Journal of Zhejiang Normal University:Natural Sciences

基　　金：国家自然科学基金资助项目(30872254);浙江省科技厅公益基金资助项目(2010C33174)

摘　　要：为了检测细胞周期性蛋白激酶CDK1与CDK2干扰对CBRH-7919细胞周期的影响,构建了CDK1和CDK2特异性sh RNA慢病毒沉默表达载体,三质粒共转染293FT细胞产生病毒颗粒,收集浓缩后感染CBRH-7919细胞,荧光显微镜下观察了细胞形态,实时定量荧光PCR和聚丙烯酰胺凝胶电泳检测了细胞中CDK1和CDK2 m RNA和蛋白质表达水平的变化,MTT法和流式细胞仪分别检测了细胞增殖和细胞周期的变化情况.结果表明:成功构建了CDK1与CDK2特异性sh RNA慢病毒表达载体,干扰CDK1导致细胞G2/M期的阻滞,细胞增殖明显降低,细胞碎片增多;而干扰CDK2后细胞仍正常生长.In order to investigate the influence of CDK1 and CDK2 interference on cell cycle in CBRH-7919 cell,the CDK1,CDK2 specific sh RNA lentiviral expression vectors were structured,then three plasmids were contransfected into 293 FT cells to produce viral particles,which infected the CBRH-7919 cells after collecting and concentrating the virals. The morphological changes of cells were observed by fluorescence microscope,Real-time PCR and Western Blotting demonstrated the level changes of CDK1,CDK2 m RNA and protein expression in CBRH-7919 cells. It was analyzed the changes of cell proliferation and cycle effect by MTT and flow cytometry. The results showed that it was successful to construct the CDK1 and CDK2 specific sh RNA lentiviral expression vector; silencing of CDK1 led to arrest of cells in G2/ M phase,cell proliferation rate decreased obviously,and increased cell debris,while silencing CDK2 cells remained growth as normal.

关 键 词：细胞周期依赖性蛋白激酶 细胞周期 细胞增殖 RNA干扰 慢病毒 

分 类 号：Q291[生物学—细胞生物学]