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目的探讨在p53缺失和突变型白血病细胞内PPP2R5A表达量的下降与细胞生物学变化之间的关系,为寻找核干细胞因子(NS)的非p53依赖性信号转导通路提供依据。方法利用脂质体转染技术将针对PPP2R5A的siRNA转染到p53缺失或突变型的白血病细胞HL-60、THP-1和Raji细胞内,沉默PPP2R5A基因。RT-PCR法和免疫细胞化学法检测siRNA的转染效果并找出最佳转染浓度和时间。倒置显微镜观察转染组和对照组细胞的生长状态并绘制细胞生长曲线。瑞-吉染色观察转染组和对照组细胞的胞核、染色质及形态改变。流式细胞术AnnexinV/PI双染法进行细胞凋亡分析,PIDNA染色法进行细胞周期测定。结果倒置显微镜观察显示转染后细胞生长状态和形态均出现变化,瑞-吉染色显示转染后细胞有凋亡小体形成。细胞凋亡分析和细胞周期测定显示转染组凋亡细胞百分比率增高,G1期和G2/M期细胞百分比上升,而S期细胞百分比减低,表明细胞增殖减弱。结论抑制p53缺失或突变型白血病细胞PPP2R5A基因后细胞的凋亡、分化有一定变化,提示p53缺失或突变型白血病细胞中PPP2R5A在细胞分化、凋亡调控中扮演一定角色,为验证NS非p53途径的信号通路是否与PPP2R5A有关提供了依据。
Objective To investigate the relationship between the decline of PPP2R5A expression in p53-deficient and mutant leukemia cells and the changes of cell biology, and to provide a basis for finding a non-p53-dependent signal transduction pathway of nuclear stem cell factor (NS). Methods siRNA targeting PPP2R5A was transfected into HL-60, THP-1 and Raji cells with p53 deletion or mutation by lipofection. The PPP2R5A gene was silenced. The transfection efficiency of siRNA was detected by RT-PCR and immunocytochemistry, and the best transfection concentration and time were found out. Inverted microscope was used to observe the growth of transfected and control cells and the cell growth curve was drawn. Rui-Kyrgyzstan staining of the transfected and control cells were observed in the nucleus, chromatin and morphological changes. Flow cytometry AnnexinV / PI double staining for apoptosis analysis, PIDNA staining for cell cycle determination. Results Inverted microscopy showed that the cell growth and morphology changed after transfection, and Rui-Kyr staining showed that apoptotic bodies formed after transfection. Apoptosis analysis and cell cycle assay showed that the percentage of apoptotic cells in transfected cells was increased, while the percentage of cells in G1 phase and G2 / M phase increased while the percentage of S phase cells decreased, which indicated that the cell proliferation decreased. Conclusion Inhibition of PPP2R5A gene in p53-deficient or mutant leukemic cells may change the apoptosis and differentiation of cells, suggesting that PPP2R5A may play a role in the regulation of cell differentiation and apoptosis in p53-deficient or mutant leukemia cells. Of the signal path and PPP2R5A provide the basis.