吉林大学学报(医学版)

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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

蔡文涛1,2,夏 虹3,沈宁江2,林明侠2   

  1. 1. 南方医科大学研究生学院,广东 广州510000;2.海南省人民医院骨科二区,海南 海口570311;3.广州军区总医院骨科医院骨科一区,广东 广州510010
  • 收稿日期:2014-01-08 出版日期:2014-07-28 发布日期:2015-01-18
  • 通讯作者: 夏 虹(Tel:020-88653334,E-mail: cabbage7724@126.com) E-mail:cabbage7724@126.com
  • 作者简介:蔡文涛(1977-),男,湖北省鄂州市人,主治医师,医学博士,主要从事骨肿瘤基础及临床研究。
  • 基金资助:

    海南省科技厅自然科学基金资助课题(809048)

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

CAI Wen-tao1,2,XIA Hong3,SHENG Ning-jiang2,LIN Ming-xia2   

  1. 1.Graduate School,Southern Medical University,Guangzhou 510000,China;2.Department of Orthopedics,Hainan Provincial People’s  Hospital,Haikou 570311,China;3.Department  of Orthopedics,General Hospital,Guangzhou Military     Command of PLA,Guangzhou 510010,China
  • Received:2014-01-08 Online:2014-07-28 Published:2015-01-18

摘要:

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TG2 siRNA缺氧组(转染TG2 siRNA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72 h) 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、TG2 mRNA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,TG2 siRNA缺氧组细胞各时间点TG2 mRNA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。

关键词: 骨肉瘤, Ⅱ型谷氨酰胺转氨酶, bcl-2相关x蛋白质, 细胞色素C, 细胞凋亡

Abstract:

Abstract: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 72 h) 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.

Key words: osteosarcoma, transgiutaminase 2, bcl-2-associated x protein, cytochrome C, apoptosis

中图分类号: 

  • R738.1