抑制p53缺失和突变型白血病细胞PPP2R5A表达后细胞生物学变化

Cytobiological effect on the leukemia cells with p53 deletion and mutation by inhibiting PPP2R5A expression

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 explore the cytobiological effect on the leukemia cells with p53 deletion and mutation by inhibiting PPP2R5A expression,laying a foundation of finding the signal transduction pathway independently on p53.Methods Transfected siRNA-PPP2R5A to the leukemia cells HL-60,THP-1 and Raji by liposome transfect technology to silent the PPP2R5A.Test the transfect effect by RT-PCR and immunocytochemistry to find the best concentration of siRNA-PPP2R5A and transfect time.Observe the cells growth by inverted microscope.Observe the cells form by Wright-Giemsa dye.Test the change of cell apoptosis and cell cycle by FCM.Results Under the inverted microscope,the cells' growth state become different.Wright-Giemsa dye showed that the cells form to apoptotic body.When we analysis the result of cell apoptosis and cell cycle,we find the apoptosis cells in treatment group were more than control groups,wend the cells at G1 phase and G2/M phase in treatment group were more than control groups,while the cells at S phase were less than control groups,which indicates that the proliferation become weak.Conclusions We test and verify the relation between PPP2R5A and cell apoptosis and differentiation,laying a foundation of finding the signal transduction pathway independently on p53.