Ⅱ型谷氨酰胺转氨酶对骨肉瘤MG-63细胞凋亡的抑制作用及其机制

Inhibitory effect of transgiutaminase 2 on apoptosis of osteosarcoma cell line MG-63 and its mechanism

目的:研究骨肉瘤MG-63细胞中Ⅱ型谷氨酰胺转氨酶(TG2)的抗凋亡作用,探讨其抑制肿瘤细胞凋亡的机制。方法:设计针对TG2的siRNA,建立骨肉瘤MG-63细胞体外缺氧培养模型,分为常氧组(细胞在常氧下培养)、单纯缺氧组(细胞在缺氧培养箱里培养)、对照siRNA缺氧组(转染对照siRNA后在缺氧培养箱里培养)和TG2siRNA缺氧组(转染TG2siRNA后在缺氧培养箱里培养)。观察各组骨肉瘤MG-63细胞在缺氧培养不同时间(6、12、24、48和72h)后Bax和Cyt C的表达及细胞凋亡率。微量滴定法检测TG2活性,RT-PCR法检测TG2和Bax mRNA表达水平,免疫组织化学法检测TG2和Bax蛋白在细胞内的分布,Western blotting法检测TG2、Bax和Cyt C蛋白表达水平,流式细胞术检测细胞凋亡率。结果:与常氧组比较,单纯缺氧组和对照siRNA缺氧组细胞TG2活性、TG2mRNA和蛋白表达水平明显增强(P<0.01),并随缺氧时间延长逐渐升高(P<0.01);Bax mRNA表达水平变化不明显(P>0.05),但Bax蛋白表达水平明显下降(P<0.01),细胞胞浆和线粒体内Cyt C蛋白水平及细胞凋亡率无明显变化(P>0.05)。与单纯缺氧组和对照siRNA缺氧组比较,TG2siRNA缺氧组细胞各时间点TG2mRNA和蛋白表达水平明显降低(P<0.01),Bax mRNA表达水平无明显变化(P>0.05),Bax蛋白表达明显增加(P<0.01),胞浆内Cyt C蛋白水平明显增加(P<0.01),线粒体内Cyt C蛋白水平明显降低(P<0.01),细胞凋亡率显著增加(P<0.01)。结论:TG2通过与Bax形成混合物而消耗Bax,阻止Cyt C释放至胞浆,从而抑制肿瘤细胞凋亡。

Objective To investigate the anti-apoptosis effect of transgiutaminase 2（TG2） in osteosarcoma cell line MG-63 and to explore the mechanism of inhibiting apoptosis of tumor cells.Methods The TG2-tgrgeted siRNA was designed,and the hypoxia culture model of MG-63 cells was established by a hypoxia incubator and the cells were divided into four groups：normal oxygen group,the cells were cultured under normal oxygen;hypoxia group,the cells were cultured in hypoxic incubators;control siRNA hypoxia group,the cells were cultured in hypoxic incubators after transfection of control siRNA;TG2 siRNA hypoxia group,the cells were cultured in hypoxic incubators after transfection of TG2 siRNA.The expressions of Bax and cytochrome C（Cyt C）and the apoptotic rates were observed at different time（6,12,24,48,and 72h）after hypoxia culture.Microtiter plate assay was performed to monitor the intracellular TG2 activity.RT-PCR was used to detect the mRNA expressions of TG2 and Bax.The expressions levels of TG2,Bax and Cyt C were observed by immunohistochemical staining and Western blotting method.The apoptotic rates were analyzed using flow cytometry.Results Compared with normal oxygen group,the activity of TG2,the mRNA and protein expression levels of TG2 in hypoxia group and control siRNA hypoxia group were increased remarkably with the prolongation of the hypoxia time（P〈0.01）;the expression level of Bax protein was decreased significantly（P〈0.01）,but the expression level of Bax mRNA had no significant change（P〈0.05）;the expression levels of Cyt C protein in cytoplasm and mitochondria and the apoptotic rates had no markedly changes（P〈0.05）.Compared with hypoxia group and control siRNA hypoxia group,the expression levels of mRNA and protein of TG2 in TG2 siRNA hypoxia group were decreased significantly at different time points（P〈0.01）;the protein expression levels of Bax and Cyt C in cytoplasm and the apoptotic rates were increased markedly（P〈0.01）;the expression level of Cyt C in mitochondria was decreased（P〈0.01）.Conclusion TG2 can inhibit the apoptosis of tumor cells through down-regulating the Bax expression and preventing Cyt C releasing into the cytoplasm.