p53及其凋亡刺激蛋白对结肠癌细胞自噬和凋亡影响机制探讨

Research of mechanism of p53 and apoptosis stimulating protein on autophagy and apoptosis in colon cancer cell

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摘要目的探讨p53、p53凋亡刺激蛋白2(apoptosis stimulating protein 2 of p53,ASPP2)及异构体△ASPP2在p53缺陷结肠癌细胞系HCT116-/-细胞中对自噬及凋亡的影响.方法实验分为对照组、MMS组、MMS+ p53组、MMS+ASPP2+ p53组、MMS+ △ASPP2+ p53组和MMS+ ASPP2+△ASPP2+ p53组.应用ASPP2、△ASPP2及p53质粒转染HCT116-细胞,同时转染绿色荧光GFP-LC3质粒于各组细胞中,经甲基磺酸(MMS)处理后用荧光显微镜观察细胞自噬水平,蛋白质印迹法检测各组细胞自噬及凋亡相关蛋白表达水平,Calcein AM/PI染色检测细胞凋亡水平.结果自噬水平对照组为(0.63±0.14)％,MMS组为(10.48±0.58)％,MMS+ p53组为(5.45±0.41)％,MMS+ ASPP2+p53组为(3.43±0.35)％,MMS+ △ASPP2+ p53组为(14.84±0.64)％,MMS+ ASPP2+ △ASPP2+ p53组为(12.29±0.58)％,差异有统计学意义,F=1 182.364,P＜0.001;与MMS组比较,MMS+ p53组和MMS+ ASPP2+ p53组自噬水平下降,P值均＜0.001;与MMS+ p53组相比,MMS+ ASPP2+ p53组自噬水平降低,P＜0.001;MMS+△ASPP2+p53组自噬水平较MMS+ p53处理组增加,P＜0.001;MMS+ ASPP2+△ASPP2+ p53组自噬水平较MMS+ ASPP2+p53组增加,P＜0.001.凋亡率比较,对照组为(2.09±0.54)％,MMS组为(16.79±0.69)％,MMS+p53组为(33.70±1.16)％,MMS+ ASPP2+ p53组为(50.61±1.17)％,MMS+△ASPP2+ p53组为(21.32±1.18)％,MMS+ ASPP2+△ASPP2+ p53组为(35.22±1.06)％,差异有统计学意义,F=2 571.942,P＜0.001;MMS+ p53和MMS+ ASPP2+ p53组凋亡较MMS组增加,P值均＜0.001;与MMS+p53组相比,MMS+ ASPP2+ p53组凋亡增加,P＜0.001;MMS+△ASPP2+ p53组凋亡较MMS+ p53组减少,P＜0.001;MMS+ ASPP2+△ASPP2+ p53组凋亡较MMS+ ASPP2+ p53组也有所降低,P＜0.001.结论 ASPP2能够增强p53的自噬抑制作用,并增强p53促细胞凋亡作用,△ASPP2能够抑制ASPP2与p53的功能,具有促进细胞自噬,抑制细胞凋亡的作用.