LPS通过调节Cdc2蛋白表达诱导HeLa细胞凋亡的研究

LPS induces HeLa cells apoptosis by regulating expression of Cdc2 protein

目的构建细胞分裂周期蛋白2(Cdc2)真核表达载体,并在P16基因缺失的肿瘤细胞HeLa细胞中表达,利用LPS诱导细胞凋亡,并探讨LPS诱导HeLa细胞凋亡的机制。方法从人U2OS细胞中提取RNA,反转录克隆Cdc2cDNA片段,利用Gateway技术构建pDEST27-Cdc2质粒。将pDEST27-Cdc2质粒瞬时转染人宫颈癌HeLa细胞,免疫印迹分析Cdc2的表达情况,并用LPS刺激,观察其对LPS引起的细胞凋亡和细胞周期的影响。结果成功构建pDEST27-Cdc2质粒,质粒酶切及测序结果完全正确。LPS刺激HeLa细胞24h后,流式细胞仪检测发现大量细胞凋亡,而转染pDEST27-Cdc2质粒的细胞凋亡率明显降低,提示Cdc2的重要作用。免疫印迹分析研究发现,LPS诱导HeLa细胞Cdc2蛋白水平下降,进一步研究发现LPS诱导HeLa细胞G2/M期阻滞,过表达Cdc2可以降低G2期细胞阻滞的发生。结论 LPS诱导HeLa细胞凋亡是由于其导致Cdc2表达下降,细胞分裂停止在G2/M期,过表达Cdc2可以阻止死亡信号诱导的细胞凋亡。

Objective To construct the ukaryotic vector of cell division cycle 2（Cdc2）,to express it in the HeLa cells which lack p16genes,to induce cell apoptosis by using LPS,and to investigate the apoptosis mechanism of HeLa cells induced by LPS.Methods Cdc2 cDNA fragments were cloned from human U2OS cells by reverse transcription,the pDEST27-Cdc2 plasmid was constructed by Gateway technology.To transiently transfect the pDEST27-Cdc2 plasmid into the HeLa cells by liposome.The expression of Cdc2 was analyzed by Western blot and its influence on the cell cycles and cell apoptosis stimulated by LPS was observed.Results pDEST27-Cdc2 plasmid was constructed successfully.Restriction analysis and sequencing proved the same as the design.After 24 h stimulation on HeLa cells by LPS,a large number of apoptosis cells were found by the flow cytometry,the apoptosis of HeLa cells transfecting pDEST27-Cdc2 plasmid was significantly decreased,which prompted the important role of Cdc2.Western blot study found that the expression of Cdc2 in HeLa cells stimulated by LPS was decreased.Further study found that LPS could induce the G2/M phase retardation in HeLa cells.And overexpression of Cdc2 could reduce the occurrence the G2/M phase retardation. Conclusion LPS induces the apoptosis of HeLa cells by down-regulating the expression of Cdc2 protein and stopping the cell divi-sion in G2/M phase.Overexpression of Cdc2 can prevent the cell apoptosis induced by death signal.