p53 ser249突变对小鼠EF细胞周期和凋亡的影响  

Effect of p53 249 Arg to Ser mutation on cell cycle and apoptosis in mouse EF cells

作　　者：吴智群[1] 李庆霞[1] 李臻[1] 于翠娟[1] 罗亚宁[1] 

机构地区：[1]第四军医大学唐都医院介入科,西安710038

出　　处：《中华肝胆外科杂志》2009年第10期761-763,共3页Chinese Journal of Hepatobiliary Surgery

基　　金：国家自然科学基金（30572101）

摘　　要：目的研究p53 249编码子突变对小鼠EF细胞p53功能和信号传导的影响。方法利用基因打靶技术在小鼠ES细胞p53基因249编码子中引入点突变，使编码子249由Arg变成Ser，根据同源重组规律采用PCR或Southern方法筛选带有p53 249突变的阳性ES细胞，并通过测序确定该ES细胞的p53 249编码子已经由Arg变成Ser，然后将含突变而不含筛选标记的ES细胞微注射到从Hprt-／-小鼠收集的囊胚中，将注射过的囊胚植入假孕的雌性小鼠子宫，到第14天取小鼠胚胎EF细胞，用含HAT的培养液EDMEM含10％FCS，glutamine，antibiotics，50 mM mecaptoethanol，和HAT（0．016mg of hypoxanthine／ml，0．01 mM aminopterin，0．0048mg of thymidine／m1）]筛选出从ES细胞分化而成的MEF细胞，经测序证实该细胞含有249Arg到Ser的突变后，该细胞用于研究。利用细胞流式仪检测该突变对MEF细胞周期及凋亡的影响，并利用Western检测相关蛋白的表达。结果（1）用uV处理EF细胞后，含p53249编码子突变的EF细胞凋亡百分数，较含野生型p53的EF细胞凋亡百分数明显减少（P〈0．05）。（2）用UV处理EF细胞后，含p53 249编码子突变的EF细胞对UV诱导的G1／G0细胞周期阻滞作用减弱。（3）用UV处理细胞后，含p53 249编码子突变EF细胞的p53的表达，与含野生型p53EF细胞p53的表达没有差别，但含p53249编码子突变EF细胞的BAX和p21的表达，较含野生型p53EF细胞的BAX和P21的表达减少。结论p53 249编码子突变可以减弱p53在UV诱导MEF细胞周期阻滞和凋亡过程中的作用，但对p53的表达没有影响。Objective To explore the effect of p53 249 Arg to Ser mutation on function of p53 in mouse EF cells. Methods Arg to Set mutation was introduced into the p53 249 code by knock-in method. The ES cells with p53 249 Arg to Set mutation were selected according to the homologues-recombination with RCR and Southern blot. The positive ES cells without a selection marker were injected into blastocysts recovered from Hprt-/- mice, which were derived from Hprt-deficient ES cells. The injected blastocysts then were implanted into pseudopregnant females. At embryonic day 14, MEFs were recovered from the embryos and cultured under the selection of HAT （0. 016 mg of hypoxanthine/ml, 0.01 mM aminopterin, 0. 0048 mg of thymidine/ml） and the cell cycle, apoptosis, and the relative protein expression were analyzed by flow eytometry and Western blot. Results 1） The EF cells with 249 Arg to Ser mutation were more resistant to the UV induced cell cycle arrest compared to the wild type EF cell （P〈0.05）. 2） The EF cells with 249 Arg to Ser mutation were more resistant to the UV induced apoptosis compared to the wild type ES cell （P〈0.05）. 3） The mutation did not affect the expression of p53 after UV but decrease the expression of BAX and p21 after UV. Conclusion Thep53 249 Arg to Ser mutation can disable the function of p53 but do not change the expression of p53 in EF cells.

关 键 词：癌 肝细胞 发病机制 抑癌基因P53 

分 类 号：R686[医药卫生—骨科学]