PUMA通过线粒体途径诱导骨肉瘤细胞凋亡的研究

目的:研究p53上调凋亡调控因子(PUMA)对骨肉瘤细胞凋亡的影响及机制。方法:在骨肉瘤细胞F5M2中转染PUMA过表达载体，同时转染对照阴性载体，以qRT-PCR和Western blot法测定转染后细胞中PUMA表达情况，以流式细胞术检测细胞凋亡情况，用JC-1法检测细胞线粒体膜电位，以Western blot法测定线粒体和胞浆中细胞色素C（Cyt C）蛋白表达情况，同时检测细胞中剪切的含半胱氨酸的天冬氨酸蛋白水 解酶3（Cleaved Caspase-3）、剪切的含半胱氨酸的天冬氨酸蛋白水解酶9（Cleaved Caspase-9）蛋白水平。结果:转染PUMA过表达载体后的F5M2细胞中PUMA mRNA和蛋白水平升高，而转染对照阴性载体对F5M2细胞中PUMA mRNA和蛋白水平没有影响。过表达PUMA后的F5M2细胞凋亡率升高，细胞线粒体膜电位下降，线粒体中Cyt C蛋白水平降低，胞浆中Cyt C蛋白水平升高，同时细胞中Cleaved Caspase-3、Cleaved Caspase-9蛋白水平也升高，与未做转染的F5M2细胞比，差异有统计学意义（P<0.05）。转染对照阴性载体后的细胞凋亡率、线粒体膜电位、线粒体和胞浆中Cyt C蛋白水平、Cleaved Caspase-3蛋白水平、Cleaved Caspase-9蛋白水平与未做转染的F5M2细胞相比没有明显变化（P>0.05）。结论:PUMA通过促进线粒体中Cyt C释放，降低线粒体膜电位，激活Caspase-3诱导骨肉瘤细胞凋亡。

Study on the apoptosis of osteosarcoma cells induced by mitochondrial pathway by PUMA

Objective:To study the effect of PUMA on the apoptosis of osteosarcoma cells and its mechanism.Methods:PUMA overexpression vector in osteosarcoma cell F5M2 was transfected,at the same time，the control negative vector was transfected.The expression of PUMA in the transfected cells was measured by qRT-PCR and Western blot.Cell apoptosis was detected by flow cytometry.The JC-1 method was used to detect the mitochondrial membrane potential.The expression of Cyt C protein in mitochondria and cytoplasm was measured by Western blot method.The levels of Cleaved Caspase-3 and Cleaved Caspase-9 protein were also detected.Results:The level of PUMA mRNA and protein increased in the F5M2 cells transfected with PUMA overexpression vector，the transfection control negative carrier had no effect on the level of PUMA mRNA and protein in F5M2 cells.The apoptosis rate of F5M2 cells was increased after overexpression of PUMA.The membrane potential of cell mitochondria was decreased.The level of Cyt C protein in mitochondria was reduced，and the level of Cyt C protein in the cytoplasm was increased.At the same time，the level of Cleaved Caspase-3 and Cleaved Caspase-9 protein in the cells was also increased.Compared with the F5M2 cells that were not transfected，the difference was statistically significant (P<0.05).The apoptotic rate，mitochondrial membrane potential，mitochondria and cytoplasmic Cyt C protein level，Cleaved Caspase-3 protein level and Cleaved Caspase-9 protein level of transfected control negative vectors were not significantly different from those of F5M2 cells without transfection (P>0.05).Conclusion:PUMA reduces mitochondrial membrane potential by promoting the release of Cyt C in mitochondria and activates Caspase-3 to induce apoptosis of osteosarcoma cells.