CAJ | 학술논문

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、6.25 μmol/L pk11195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、6.25 μmol/L pk11195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；Western blotting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/L pk11195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50 μmol/L pk11195组细胞死亡率、GSH水平低于对照组和6.25 μmol/L pk11195组,差异有统计学意义(P＜0.05)；6.25、50 μmol/L pk11195组细胞凋亡率、TSPO的表达量较对照组降低,且50μmol/L pk11195组低于6.25 μmol/Lpk11195组,差异有统计学意义(P＜0.05)；6.25、50 μmol/Lpk11195组ATP的含量高于对照组,且50 μmol/Lpk11195组高于6.25 μmol/L pk11195组,差异有统计学意义(P＜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.
목적 이용상대분자질량위18000적전위분자단백(TSPO)적특이성배체1-(2-록분기)-N-(1-갑기병기)-이규람-3-안갑선(pk11195)연구TSPO대인뇌효질류U251증식적영향급궤제,위인뇌효질류적치료심조신적파점. 방법 체외상규배양U251세포,응용100、50、25、12.5、6.25 μmol/L pk11195작용2h후MTT비색법검측세포적증식활성,동시설대조조(가입등량용제)；태반람염색법검측대조조화50、6.25 μmol/L pk11195조세포사망솔적변화；Hoechst33342염색법화류식세포의검측세포조망；Western blotting화면역형광법검측세포TSPO적표체；2’,7’-이경이록형광황쌍을산납(DCFH-DA)탐침법화GSH시제합분별검측세포내활성양(ROS)화GSH적수평；곡불흉감(JC-1)염색법검측선립체막전위；형광소매법검측세포내ATP함량. 결과 MTT검측현시50、25μmol/L pk11195조세포존활솔고우대조조,차이유통계학의의(P＜0.05)；태반람염색현시50 μmol/L pk11195조세포사망솔、GSH수평저우대조조화6.25 μmol/L pk11195조,차이유통계학의의(P＜0.05)；6.25、50 μmol/L pk11195조세포조망솔、TSPO적표체량교대조조강저,차50μmol/L pk11195조저우6.25 μmol/Lpk11195조,차이유통계학의의(P＜0.05)；6.25、50 μmol/Lpk11195조ATP적함량고우대조조,차50 μmol/Lpk11195조고우6.25 μmol/L pk11195조,차이유통계학의의(P＜0.05).pk11195조ROS수평교대조조강저,이세포막전위증고. 결론 TSPO구유촉진U251세포조망、억제기증식、유사우억암기인적작용.
Objective To study the effect of the 18kDa translocator protein (TSPO) on U251cells of human glioma. Methods U251 cell line was cultured in vitro conventionally.The specific ligand ofTSPO,pk11195,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.MTr 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 pk11195than in the control group (P＜0.05). Trypan blue staining showed the cell death rate was significantlylower in the group of 50 μmol/L pk11195 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 μmol/L pk11195,compared with the control group; the apoptosis rate was significantly lower in the group of 50 μmol/Lpk11195 than in the group of 6.25 μmol/L pk11195 (P＜0.05).The cell membrane potential and ATP quantity were significantly higher in the groups of 6.25 and 50 μmol/L pk11195 than in the control group,and those in the group of 50 μmol/L pk11195 were significantly higher than in the group of 6.25 μmol/Lpk11195 (P＜0.05). Conclusion TSPO may promote apoptosis of U251 cells in human glioma and inhibit proliferation of glioma cells,functioning similarly as a cancer suppressor gene.