高同型半胱氨酸诱导人成骨肉瘤MG63细胞损伤的机制  

Homocysteine-induced oxidant injury of human osteosarcoma MG63 cells and its mechanisms

作　　者：刘承杏[1] 陈学秋 郭家智[1] 戴纪男[1] 孙俊[1] 桂莉 陆地[1] 

机构地区：[1]昆明医学院人体解剖学教研室,昆明650500 [2]云南省中医学院人体解剖学教研室,昆明650500 [3]云南省第三人民医院内分泌科,昆明650011

出　　处：《解剖学报》2011年第3期356-360,共5页Acta Anatomica Sinica

基　　金：云南省应用基础研究重点项目(2008CC007);云南省中青年学术和技术带头人后备人才培养项目(2009CI033)

摘　　要：目的探讨高同型半胱氨酸(HHcy)诱导人成骨肉瘤MG63细胞损伤的分子机制。方法体外培养的人成骨肉瘤MG63细胞经不同浓度(0.001mol/L、0.003mol/L和0.005mol/L)的Hcy处理不同时间后,采用2',7'-二氯二氢荧光素二乙酸盐(H2 DCFDA)探针在荧光分光光度计下检测细胞内活性氧(ROS)变化水平;通过Hoechst 33342/碘化丙啶(PI)核双染法在荧光显微镜下观察计数凋亡和坏死细胞;采用Western blotting法检测促凋亡相关蛋白Caspase-3和Bax的表达变化。结果在高浓度的Hcy刺激下,以剂量和时间依赖的方式上调MG63细胞内ROS含量(P<0.05);同时,凋亡相关蛋白Caspase-3和Bax的表达明显增加,从而诱导MG63细胞的死亡。结论高浓度Hcy可通过增加MG63细胞内ROS的含量,上调凋亡相关蛋白Caspase-3和Bax的表达,导致MG63细胞的氧化损伤。Objective The present study is designed to examine the responses of human osteosarcoma MG63 cells to hyperhomocysteinemia（HHcy） and the possible mechanisms. Methods MG63 cells were cultured in DMEM contain 10% fetal bovine serum at 37℃ with a humidified atmosphere 5% CO2. They were treated in different concentration and different time by HHcy. MG63 ceils were used as in vitro model to assess the cell intracellular reactive oxygen species （ROS） that was evaluated by the fluorescence probe 2′,7′-dichlorodihydrofluorescein diacetate （H2DCFDA）. The cell apoptosis was observed by means of Hoechst/PI nuclear staining and fluorescence microscopy. The expressions of apoptosisrelated protein Caspase-3 and Bax were analyzed with Western blotting. Results After MG63 cells were treated with HHcy in different concentrations and for different time. HHcy induced the cell death through accumulation of intracellular ROS in a dose-and time-dependent manner, at the same time activation of Caspase-3 and upregulation of Bax apoptosis-related protein. Conclusion HHcy exerts apoptosis-inducing effects on human osteosarcoma MG63 cells through accumulation of intracellular ROS and activation of Caspase-3 and upregulation of Bax protein,

关 键 词：MG63细胞 同型半胱氨酸 活性氧 荧光探针 Hoechst 33342/PI核双染法 免疫印迹法 

分 类 号：R322.7[医药卫生—人体解剖和组织胚胎学]