慢病毒介导 CDK1／CDK2干扰对肿瘤细胞周期的影响❉

摘要

In order to investigate the influence of CDK1 and CDK2 interference on cell cycle in CBRH-7919 cell, the CDK1, CDK2 specific shRNA 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 mRNA and protein ex-pression 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 shRNA lentiviral expression vector;silencing of CDK1 led to arrest of cells in G2/M phase, cell proliferation rate de-creased obviously, and increased cell debris, while silencing CDK2 cells remained growth as normal.%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shRNA慢病毒沉默表达载体，三质粒共转染293 FT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 mRNA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shRNA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。