野生型p53基因对白血病K562细胞的增殖抑制作用

目的:建立携野生型p53基因的K562细胞株,研究该细胞内野生型p53基因的表达并观察其生物学特性。方法:构建重组人p53逆转录病毒载体,导入PA317细胞中进行包装,经过病毒滴度测定,筛选高滴度的产病毒细胞克隆。收集培养上清液中的重组人p53逆转录病毒颗粒,感染K562靶细胞,建立携野生型p53基因的K562细胞株。观察该细胞株的生长方式、形态学和生长速度。SP免疫组化染色法检测p53蛋白表达。进一步用流式细胞仪检测p53蛋白表达率和细胞周期变化;双层软琼脂培养实验检测细胞的增殖潜能改变。以pBabe空载体同时实验,作为各阶段的对照。结果:成功地构建了重组逆转录病毒载体pBabe/p53;获得逆转录病毒生产PA317/p53细胞系,最高病毒滴度为4.64×105CFU/m l;获稳定表达p53基因的K562/p53靶细胞,免疫组化结果证实该细胞内有野生型P53蛋白表达,流式细胞仪检测到野生型P53蛋白表达率为1.32%;与对照K562/pBabe细胞比较,K562/p53靶细胞的生长速度下降;软琼脂集落生成的下降率为27.14±4.49%(P<0.05);增殖指数下降,S期比例下降(P均<0.01)。结论:制备了携p53基因的高滴度逆转录病毒,建立携野生型p53基因的K562/p53细胞株,证实表达的野生型p53蛋白对该细胞有抑制作用。

Expression and Growth Suppression Effect of Exogenus Wild Type p53 Gene Induced by pBabe Retrovirus Vector in Leukemic K562 Cell Line

Objective:To establish the liukemic K562 cell line with exogenous wild type p53 gene and explore expression and biological characteristic of p53 gene.Methods:Recombinant retrovirus vector Pbabe/P53 encoding wild-type p53 gene was constructed and transformed into the cultured PA317 packaging cell line.The vector pBabe was also transformed into the cultured PA317 as the control.The positive clones were amplifyied after selectin with puromycin.The supernatant virus was used to infect Hela cells and titrated.The supernatant virus from the highest productive PA317/p53 and PA317/pBabe clones was used to infect the K562 cell line.The positive clones of K562/P53 and K562/pBabe were selected by puromycin and amplifyied.The expression of p53 protein and cell cycle of transgenic cells was tested by both SP immuneohistochemistry stain and flow cytometry.The prolixferation of transgenic cells of K562/P53 and K562/pBabe were tested by soft agar culture.Results:Recombinant retrovirus pBabe vector encoding wild type p53 gene was constructed,and the vector was packaged into PA317 cells.Highest titre of the virus from suoernatant was 4.64x105 CFU/ml.K562/p53 cells were partial positive by SP immuneohistochemistry stain.The expression of p53 protein in K562/p53 cells was 1.32% by flow cytometry analysis.The transgenic cells K562/P53 grow slower than the K562/pBabe control cells.The colony formation of K562/P53 cells was dropped 27.14 4.49% as compared to K562/ pBabe control cels revealed by soft agar test results.The K562/P53 transgenic cells at S phase of cell cycle were decreased to 42.05% as compared to 52.38% in K562/ pBabe control cells.Conclusion:The cell line K562/053 could be established by transfercting wild type p53 gene effectively into K562 cells with pBabe retrovirus vector,growth of K562/P53 cells stably expressed human wild type p53 gene,therefore these cells was suppressed by wild type p53 gene.