p53 249突变Knock-in动物模型的建立及其在肝癌发病中的作用

A mouse model of p53 249 Knock-in mutation and its role in liver carcinogenesis

目的:观察p53249突变在肝细胞癌变发生诱导的作用。方法:利用基因打靶技术在小鼠胚胎干细胞(ES)中引入p53249编码子Arg(精氨酸)至Ser(丝氨酸)突变,然后经显微注射到小鼠囊胚中,并将注射后的囊胚植入假孕小鼠的子宫。得到嵌合小鼠后与BL/6小鼠杂交得到p53249Knock-in小鼠。观察分析该小鼠的生长及肝脏肿瘤发生情况及病理改变。同时用DEN(二乙基亚硝胺)分别处理及饲养含249突变的Knock-in小鼠和不含突变的野生型小鼠,观察比较两组小鼠的生长情况,30周后处死小鼠,研究两组小鼠肝脏癌变情况及病理改变。结果:p53249编码子突变小鼠的生长和野生型小鼠生长没有明显差别。观察一年时间解剖小鼠,未见小鼠肝脏有肿瘤发生。当使用DEN处理和饲养两组小鼠时,突变组小鼠15周以后出现不同程度的饮食减退,精神不佳,体重下降等表现,而对照组小鼠无明显异常。30周处死小鼠发现90%(18/20)的突变组小鼠有肝脏肿瘤发生,病理显示为肝细胞肝癌,同时伴有肝硬化表现。而对照组小鼠仅5%(1/20)发现有肝脏肿瘤发生。结论:p53249编码子突变在肝癌发病过程中起着非常重要的作用。

Objective：To observe if the liver can develop hepatoma after p53 harbors the 249 code mutation. Methods： Introduce an Arg to Ser mutation in 249 code of p53 with knock - in method in mouse embryonic stems （ES） cells, and after microinjection of the positive ES into blastocyst, implant the blastocyst inside the uterus of pseu- dopregnant female mouse. After get the chimeric mouse, mate with female black 6 mouse, and get the germline 249 knock - in mouse. Observe the growth and activity of these mice, and dissect 10 of one - year - old mouse, observe the pathologic changes of mouse liver. Resluts： After introduce 249 Arg to Ser mutation into the mouse p53 ,we did not observe any difference in growth and activity between the mutated mouse and wild type mouse. And we also did not find the liver cancer happened after one year observasion. When treated the mutated and wild type mouse with DEN （ Diethylnitrosamine）, we found the mutated mouse had some varying degree the diet decrease, body weight dropping, and the state of mind was not good after 15 weeks. When dissected the mouse at 30 weeks,90% （18/20） of the mutated mouse harbored tumors with some liver cirrhosis changes,but only 5% （1/20） wild type harbored tumors. Conclusion： p53 249 mutation is playing the very vital role in liver carcinogenesis.