CDK1 siRNA干扰在Taxol诱导细胞凋亡中的作用

目的研究转染细胞周期依赖性蛋白激酶1（cyclin．dependent kinase1，CDK1）siRNA、以及转染后进行凋亡刺激对细胞周期和凋亡的影响，探讨CDK1在细胞凋亡中的确切作用，揭示细胞周期与细胞凋亡协调的分子机制。方法以人宫颈癌细胞株HeLa细胞为研究对象，脂质体转染CDK1siRNA，转染后48h加紫杉醇（Tax01）（20μg／m1）刺激凋亡，Western印迹检测CDK1和抗凋亡蛋白BCL2表达，AnnexinV／PI法检测细胞的凋亡，流式细胞仪分析DNA含量检测细胞周期。结果转染CDK1 siRNA后，CDK1蛋白的表达下降，细胞周期G2／M期比例增加，细胞凋亡率与对照相比没有明显升高。只加Taxol刺激12h后细胞凋亡率增加并伴有S期和G2／M期比例增加。转染CDKlsiRNA后再用Taxol刺激，其细胞凋亡率没有明显改变，G2／M期阻滞效应也没有叠加。BCL2蛋白只在加Taxol刺激组表达下降，与CDK1表达减少没有相关性。结论siRNA沉默导致的CDK1表达降低只导致细胞周期G2／M期阻滞，没有引起细胞凋亡；CDK1的表达降低对紫杉醇所诱导的细胞周期阻滞和细胞凋亡效应没有明显影响。

Role of CDK1 siRNA Interference in Taxol Induced Apoptosis

Objective The family of cyclin-dependent kinase complexes （Cdks） are well known for their role in cell cycle. However, the precise role of Cdks in apoptosis still remains to be defined. In the present report, we sought to understand the influence of CDK1 on cell cycle and ap- optosis in Hela cells by silencing CDK1 gene expression with CDK1 siRNA. Method The siRNA targeting CDK1 gene was chemically synthesized and transfected into Hela cells by lipo- fectamine2000. To stimulate apoptosis induction, 20 ug/ml taxol was added at 48 hour after trans- feetion. The cell cycle of the transfected cells with and without drug treatment was detected by flow cytometry. Apoptosis induction was determined by Annexin V/PI technique. Cell lysates were subjected to Western blot analysis for detection of CDK1 and Bcl2 protein expression. Result CDK1 was successfully silenced by CDK1 siRNA, as its protein expression level was significantly decreased. Transfection of CDK1 siRNA led cells to G2/M phase but did not trigger cell apoptosis. Taxol treatment （20ug/ml） induced obvious cell apoptosis and cell-cycle arrest in S and G2/M phase as early as 12h after drug addition, whereas CDK1 siRNA did not affect the degree of cell cycle arrest and apoptosis. Furthermore, the anti-apoptotic Bcl-2 protein was decrease when apoptosisinducing drug taxol was added, but remained constant after CDK1 siRNA transfection. Conclusion The decreased CDK1 expression by gene silence induced cell cycle arrest but not apoptosis. The apoptosis and cell cycle arrest triggered by apoptotic stimulating treatment was not influenced by CDK1 siRNA.