PPI1诱导人宫颈癌HeLa细胞G2／M期停滞及机制研究  

The mechanism of G_2/M arrest induced by PPI1 in human cervical carcinoma HeLa cells.

作　　者：曾麒燕[1] 黄毓[2] 张红[1] 

机构地区：[1]广西医科大学生物化学与分子生物学教研室,南宁530021 [2]广西医科大学第一附属医院临床医学实验中心

出　　处：《现代妇产科进展》2008年第4期259-263,共5页Progress in Obstetrics and Gynecology

基　　金：广西自然科学基金资助课题(No:0542077)

摘　　要：目的:探讨Ⅰ型磷酸酶抑制亚基1(PPI1)对人宫颈癌细胞系HeLa细胞周期的影响及其作用机制。方法:分别将PPI1野生型和短片段突变活化型基因通过脂质体介导转染人宫颈癌HeLa细胞,用RT-PCR和Western blot鉴定目的基因的表达;用流式细胞术分析瞬时转染HeLa细胞的细胞周期。对稳定转染的HeLa细胞用脱氧胸苷处理,通过流式细胞术观察PPI1对同步化HeLa细胞的细胞周期进展的影响。用免疫印迹分析细胞周期相关蛋白表达水平的变化。结果:野生型和突变活化型PPI1在HeLa细胞中均能有效表达。突变活化型PPI1基因的瞬时转染可使HeLa细胞G2/M期比例明显升高。经脱氧胸苷处理过的同步化的稳定转染的HeLa细胞中,突变活化型和野生型PPI1均可使HeLa细胞停滞在G2/M期的时间长于对照组,而且前者更明显。免疫印迹显示:突变活化型PPI1瞬时表达48h后,cyclin A、cyclin B1、p53和p21表达上调。经脱氧胸苷同步化并恢复正常培养基后,稳定表达突变活化型PPI1的细胞cyclin B1在10.5和12h,p53在6、7.5、9和10.5h表达明显增强;稳定表达野生型PPI1的细胞cyclin B1在10.5h的表达也比对照组明显增强。结论:突变活化型的Ⅰ型磷酸酶抑制亚基1可诱导宫颈癌HeLa细胞G2/M期停滞,它与cyclin A和cyclin B1表达上调及cyclin B1降解滞后有关,而G2/M期停滞的维持与p53和p21表达上调有关。Objective： To explore the effect of inhibitor-1 of protein phosphatase （PPI1） on cell cycle of human cervical carcinoma HeLa cells, and its potential mechanisms. Methods：Plasmids containing wild type or activated mutant PPI1 gene（ PPI1/wt, PPI1/sI1 ） were transfected into HeLa cells by lipofectamine. The expression of PPI1/wt fusion protein and PPI1/sI1 was determined by RT-PCR and western blot. Cell cycle of transient transfected HeLa cells was analyzed by flow cytometry. For stable transfected HeLa cells, they were treated with thymidine to obtain the synchronous cells, moreover,the cell cycle progression of these synchronous cells was observed through flow cytometry. Correspondently, the expression of cell cycle regulatory proteins were determined by immunobloting. Results：PPI1/wt or PPI1/sI1 was efficiently expressed in HeLa cells respectively. In transient transfected HeLa cells with activated mutant PPI1, the proportion of cells in G2/M phase was higher than control. In synchronous stably transfected HeLa cells, the result of FACS suggested that there was no difference in G2/M entry among the groups after released from thymidine arrest, but both of activated mutant and wild type of PPI1 could delay mitotic exit, especially the former had longer G2/M phase. Western blot showed that the level of cyclin A, cyclin B1, p53 and p21 were up-regulated in those cells transient expressed activated mutant of PPI1. In PPI1/sI1 stably transfected HeLa cells, after released from thymidine arrest,cyclin B1 was up-regulated in 10.5h and 12h,as well as p53 in 6h,7.5h,9h and 10.5h. In stably PPI1/wt transfected HeLa cells,cyclin B1 was also up- regulated in 10. Sh. Conclnsions：The results indicated that the activated mutant PPI1 can induce HeLa cells G2/M arrest, which is related to the up-regulated expression of cyclin A and cyclin B1, as well as the delayed degradation of cyclin B1, and the sustaining of G2/M arrest involved the up-regulated expression of p53 and p21.

关 键 词：宫颈肿瘤 Ⅰ型磷酸酶抑制亚基-1 G2/M期停滞 细胞周期蛋白质类 HELA细胞 

分 类 号：R737.33[医药卫生—肿瘤]