P53 ser249突变对小鼠ES细胞细胞周期和凋亡的影响

The effect of p53 249 Arg to Ser mutation on the cell cycle and apoptosis in mouse ES cell

目的研究p53249编码子突变对p53结构、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杂交方法,筛选带有p53249突变的阳性ES细胞,并通过测序确定该ES细胞的p53249编码子已经由Arg变成Ser。利用细胞流式仪检测该突变对细胞周期及凋亡的影响,并利用Western印迹检测相关蛋白的表达。结果①用IR处理细胞后,含p53249编码子突变的ES细胞凋亡百分数,较含野生型p53的ES细胞凋亡百分数明显减少,P<0.05。②用IR处理细胞后,含p53249编码子突变的ES细胞对IR诱导的G1/G0细胞周期阻滞作用减弱。③与含野生型p53的ES细胞相比,用IR处理细胞后,含p53249编码子突变ES细胞的p53的表达没有差别,但其BAX和p21的表达,较含野生型p53的ES细胞少。结论p53249编码子突变可使p53的功能减弱,但对p53的表达没有影响。

[ Objective ] To explore the effect of p53 249 Arg to Ser mutation on the cell cycle arrest and apoptosis induced by IR in mouse ES cell. [Methods ] Arg to Ser mutation were introduced into the p53 249 code by knock-in method, the ES cell with p53 249 Arg to Ser mutation were selected according to the homologues-reeombination with PCR and Southern blot. The positive ES were treated with IR, and the cell cycle, apoptosis, and the relative protein expressions were analyzed by flow cytometry and Western blot. [ Results ] （1）The ES cell with 249 Arg to Ser mutation were more resistant to IR induced cell cycle arrest compared to the wild type ES cell, P 〈0.05. （2）The ES cell with 249 Arg to Ser mutation were more resistant to the IR induced apoptosis compared to the wild type ES cell, P 〈0.05. （3）The mutation did not effect the expression of p53 after IR, but decreased the expression of BAX and p21 after IR. [Conclusion] The p53 249 Arg to Ser mutation can disable the function of p53, but do not change the expression of p53.