LPS通过调节Cdc2蛋白表达诱导HeLa细胞凋亡的研究
赵丽君;周辉;赵明
【期刊名称】《重庆医学》 【年(卷),期】2014(000)016
【摘要】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.%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 Cdc2 cDNA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24 h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。 【总页数】4页(2012-2015)
【关键词】细胞周期蛋白质类;周期素依赖性激酶1;细胞凋亡;细胞周期 【作者】赵丽君;周辉;赵明
【作者单位】南方医科大学病理生理教研室/广东省休克微循环重点实验室，广州 510515;南方医科大学病理生理教研室/广东省休克微循环重点实验室，广州
510515;南方医科大学病理生理教研室/广东省休克微循环重点实验室，广州 510515
【正文语种】中文 【中图分类】 【文献来源】
https://www.zhangqiaokeyan.com/academic-journal-cn_chongqing-medicine_thesis/0201231355685.html 【相关文献】
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LPS通过调节Cdc2蛋白表达诱导HeLa细胞凋亡的研究
