摘要
目的 利用四环素负调控表达载体诱导表达鼠野生型p5 3、假野生突变型p5 3和突变型p5 3(172位点氨基酸分别是精氨酸、亮氨酸和组氨酸 ) ,比较一个位点不同DNA结构的p5 3minigene对细胞生长的影响。方法 通过基因重组构建四环素负调控表达的小鼠上述 3种p5 3minigene真核表达载体 ,由LipofectAMINE介导转染p5 3突变的PG细胞 ,嘌呤霉素筛选得到稳定克隆 ,利用四甲基偶氮唑盐比色法、流式细胞术、蛋白质免疫印迹等方法比较 3种p5 3对细胞体外生长的影响。结果 野生型和假野生突变型p5 3可以导致细胞生长变慢、细胞周期阻滞、p2 1表达增高 ,突变型则无以上效应。结论 野生型和假野生突变型p5 3具有抑制细胞生长引起周期阻滞的功能 ,突变型p5 3则丧失了野生型p5 3的抑瘤功能 ;p5 3基因的某些突变 (如鼠 172位点精氨酸→亮氨酸 )
Objective To investigate the effect of mouse 172 wild type p53(Arg), pseudo wild mutant type p53(Arg→Leu) and mutant type p53(Arg→His) induced by absence of tetracycline on the growth of PG cell line. Methods Three variant types of p53 minigene were sub cloned by gene recombination into an expression vector which was controlled by tetracycline. Through LipofectAMINE, the vectors were transfected into p53 defective PG(248CGG→CTT) cells, and the transfectants were screened in the selecting medium containing puromycin. Tumor suppressing effects were studied by MTT absorption, flow cytometry and Western blotting. Results Wild type p53 and pseudo wild mutant type p53 could lead cells to decrease their growth rates, arrest cell cycle and transactivation of p21 WAF1 . Mutant type p53 was defective in tumor suppression. Conclusion Wild type p53 and pseudo wild type p53 may inhibit cell growth and induce cell cycle arrest. Some p53 variants such as 172 Arg→Leu can still retain the tumor suppression function of the wild type.
出处
《中华病理学杂志》
CAS
CSCD
北大核心
2000年第5期359-362,共4页
Chinese Journal of Pathology
