南蛇藤茎提取物对人胶质母细胞瘤细胞株U251增殖和凋亡的影响及机制

Effect of Celastrus orbiculatus Extract on Cell Proliferation and Apoptosis of U251 Cells and Its Molecular Mechanism

目的:探讨南蛇藤茎乙酸乙酯提取物(Celastrus orbiculatus extract,COE)抗人胶质母细胞瘤U251细胞株的增殖和凋亡作用,研究COE抗肿瘤的分子机制,为寻找新的治疗胶质母细胞瘤药物提供依据。方法:以人胶质母细胞瘤细胞株U251为研究对象,设空白组和COE组,应用噻唑蓝[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,MTT]比色法观察COE对细胞活力的抑制作用;5-溴脱氧尿嘧啶核苷(5-bromo-2-deoxy uridine,Brd U)标记检测COE对U251细胞增殖的影响;Annexin V/碘化丙啶(PI)双标法检测COE对U251细胞凋亡的作用;透射电镜下观察经COE干预后U251细胞超微结构变化;蛋白免疫印迹法(Western blot)检测COE对凋亡标志基因B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2),Bcl-2相关X蛋白(Bcl-2-associated X,Bax)和半胱氨酸蛋白酶-3(Caspase-3)蛋白表达的影响。结果:与空白组比较,COE能够明显抑制U251细胞的增殖(P<0.05);诱导U251细胞的早期凋亡,电镜下可见到凋亡小体,影响并改变U251细胞超微结构;COE能促进Caspase-3,Bax蛋白表达,降低Bcl-2,Bcl-2/Bax蛋白表达(P<0.05)。结论:COE能有效抑制胶质母细胞瘤U251细胞株的增殖,促进肿瘤细胞凋亡,其作用机制与调节Bcl-2和Caspase-3表达有关。

Objective： To observe the effect of different concentrations of Celastrus orbiculatus extract （COE） on proliferation and apoptosis of U251 cells （a glioblastoma cancer cell line）, and its possible molecular mechanism. Method： U251 cells at the logarithmic growth phase were divided into control group and COE-treated group. 3- （4, 5-dimethyl-2-thiazolyl） -2, 5-diphenyl-2-H-tetrazolium bromide （MTT） assay and 5-bromo-2- deoxy uridine （BrdU） incorporation experiment were performed to observe the ~nhibitory effect on cell viability and proliferation, respectively. Annexin V/PI double staining was used to detect the effect of COE on U251 cell apoptosis. Uhrastructural changes of U251 cells after COE treatment were observed under transmission electron microscope; the protein expressions of apoptosis marker B-cell lymphoma-2/Bcl-2-associated X （Bcl-2/Bax） and Caspase-3 were detected by Western blot. Result： Compared to control group, the proliferation of U251 cells in COE-treated group were significant inhibited by COE （P 〈 0.05）. COE could induce the early apoptosis and uhrastructure changes of U251 cells, and apoptotic bodies were observed under transmission electron microscope. The protein expressions of Bcl-2/Bax and Caspase-3 were changed in a concentration dependent manner. Conclusion： COE can effectively inhibit the proliferation of U251 cells by down-regulating the expression of Bcl-2/ Bax and increasing the protein expression of Caspase-3, and promoting the apoptosis of cell, suggesting that COE can be used as a drug candidate for treating glioblastoma.