转位分子蛋白对人脑胶质瘤U251细胞增殖的影响

Effect of the 18kDa translocator protein on U251 cells of human glioma

目的利用相对分子质量为18000的转位分子蛋白（TSPO）的特异性配体1-（2-氯苯基）-N-（1-甲基丙基）．异喹啉-3-氨甲酰（pk11195）研究TSPO对人脑胶质瘤U251增殖的影响及机制，为人脑胶质瘤的治疗寻找新的靶点。方法体外常规培养U251细胞，应用100、50、25、12．5、6．25μmol／Lpk11195作用2h后MTT比色法检测细胞的增殖活性，同时设对照组（加入等量溶剂）；台盼蓝染色法检测对照组和50、6．25μmol／Lpklll95组细胞死亡率的变化；Hoechst33342染色法和流式细胞仪检测细胞凋亡；Westernblotting和免疫荧光法检测细胞TSPO的表达；2’，7’-二氢二氯荧光黄双乙酸钠（DCFH-DA）探针法和GSH试剂盒分别检测细胞内活性氧（ROS）和GSH的水平；曲氟胸苷（JC．1）染色法检测线粒体膜电位；荧光素酶法检测细胞内ATP含量。结果MTT检测显示50、25μmol／Lpk11195组细胞存活率高于对照组，差异有统计学意义（P〈0．05）；台盼蓝染色显示50μmol／Lpk11195组细胞死亡率、GSH水平低于对照组和6．25μmol／Lpk11195组，差异有统计学意义（P〈0．05）；6．25、50μmol／Lpk11195组细胞凋亡率、TSPO的表达量较对照组降低，且50μmol／Lpk11195组低于6．25ixmol／Lpk11195组，差异有统计学意义（P〈0．05）；6．25、50μmol／Lpk11195组ATP的含量高于对照组．且50μmol／Lpk11195组高于6．25μmol／Lpk11195组．差异有统计学意义（氏0．05）。pk11195组ROS水平较对照组降低，而细胞膜电位增高。结论TSPO具有促进U251细胞凋亡、抑制其增殖、类似于抑癌基因的作用。

Objective To study the effect of the 18kDa translocator protein （TSPO） on U251 cells of human glioma. Methods U251 cell line was cultured in vitro conventionally. The specific ligand of TSPO, pkl 1195, was used in 5 experimental groups respectively with concentrations of 100, 50, 25, 12.5 and 6.25 μmol/L, in comparison with a control group. MTT colorimetry and trypan blue staining were used to detect cell proliferation. Hoechst33342 staining and flow cytometry were applied to detect cell apoptosis. Western blotting and immumofluorescence method were used to detect the expression level of TSPO. DCFH-DA probe and GSH kit were used to respectively detect the level of reactive oxygen species （ROS） and GSH level in cells. Jc-1 staining was applied to detect the mitochondrial membrane potential. Luciferase enzyme was used to detect the quantity of ATP in cells. Results MTT showed the survival of U251 cells was significantly higher in the groups of 50 and 25 μmol/L pkl 1195 than in the control group （P〈0.05）. Trypan blue staining showed the cell death rate was significantlylower in the group of 50 μmol/L pkl 1195 than in the control group （P〈0.05）. The apoptosis rate, TSPO expression, ROS and GSH levels decreased significantly in the groups of 6.25 and 50 μmo//L pkl 1195, compared with the control group; the apoptosis rate was significantly lower in the group of 50 μmol/L pk11195 than in the group of 6.25 μmol/L pk11195 （/9〈0.05）. The cell membrane potential and ATP quantity were significantly higher in the groups of 6.25 and 50μmol/L pkl 1195 than in the control group, and those in the group of 50 μmol/L pkl 1195 were significantly higher than in the group of 6.25 μmol/L pkl 1195 （P〈0.05）. Conelnsion TSPO may promote apoptosis of U251 cells in human glioma and inhibit proliferation of glioma cells, functioning similarly as a cancer suppressor gene.