p53^(301-393)突变体的细胞定位及对有丝分裂的影响  

Localization of p53^(301-393) mutant and its effect on mitosis

作　　者：徐熠熠 蓝建平[1] 朱园园[1] 余建[1] 来晓喻[1] 孙洁[1] 黄河[1] 

机构地区：[1]浙江大学医学院附属第一医院血液科,浙江杭州310003

出　　处：《浙江大学学报（医学版）》2007年第4期331-336,共6页Journal of Zhejiang University(Medical Sciences)

基　　金：国家重点基础研究发展计划(973计划)资助项目(2002CB713700);国家自然科学基金资助项目(39870339)

摘　　要：目的:研究p53N端缺失突变体(301-393氨基酸)的绿色荧光融合蛋白p53301-393-GFP在p53阳性和阴性细胞中的定位以及对有丝分裂的影响。方法:免疫印迹检测突变体融合蛋白的表达。将野生型p53-GFP和p53301-393-GFP质粒,分别转染p53阳性细胞株HeLa和p53阴性细胞株H1299,观察各融合蛋白的细胞定位。观察转染突变体后H1299细胞生长情况,计数有丝分裂期的细胞数。HeLa细胞转染突变体后,流式细胞仪检测细胞周期,免疫印迹检测p53蛋白水平的改变。结果:p53301-393-GFP融合蛋白能正确表达。突变体在p53阳性和阴性细胞中都弥散定位于细胞核,但只在p53阳性细胞中才定位于核仁。p53301-393-GFP融合蛋白对H1299细胞的有丝分裂有明显的抑制作用,使进入有丝分裂期的细胞数量明显较对照组减少,且能够诱导凋亡。转染突变体的HeLa细胞产生G2/M期阻滞(P<0.05),但是内源性p53蛋白水平无明显改变。结论:p53301-393-GFP突变体在p53阳性和阴性细胞中有不同的细胞定位,并能抑制细胞的有丝分裂,使细胞发生G2/M期阻滞。Objective： To observe the localization of p53^301-393（residues 301-393）in p53 positive/negative cells and its effect on cell mitosis. Methods： The protein expression of p53-GFP and p53^301-393-GFP was checked by immunoblotting after transfection. Immunofluorescence staining was performed to detect the localization of wide type and mutant in Hela cells （p53 positive） and H1299 cells （p53 negative）. The cell morphology of H1299 cells transfected of p53^301-393-GFP and the cells in mitotic phase were observed. Cell cycle was analyzed by flow cytometry and p53 protein level in HeLa cells was evaluated by Western blot after transfection of p53-GFP and p53^301-393-GFP. Results： The protein expression of p53-GFP and p53^301-393-GFP was verified,p53-GFP was about 90 kMr and p53^301-393-GFP about 40 kMr. Immunofluorescence microscopy demonstrated that both proteins were diffusely located in the nuclei of HeLa cells and H1299 cells. But different from the p53-GFP,the p53^301-393-GFP was distributed in the nucleolus of HeLa cells. After transfection of the two plasmids,mitosis was inhibited in H1299 cells and some cells underwent apoptosis. G2/M progression of HeLa cells could be blocked by transfection of p53^301-393-GFP, but endogenous p53 protein level was not changed. Conclusion： p53^301-393 has a different localization in the p53 positive and p53 negative cells and could inhibit mitosis and cause the cell cycle arrest in G2/M.

关 键 词：蛋白质P53 细胞周期 肿瘤细胞 培养的 基因表达 有丝分裂 H1299细胞 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]