摘要
目的：探讨链球菌蛋白诱导人脑多形性胶质母细胞瘤BT325细胞周期阻滞及凋亡的相关作用机制。方法体外培养人脑多形性胶质母细胞瘤BT325细胞，噻唑蓝（MTT）法检测细胞增殖活性，倒置显微镜下观察细胞形态变化，原位染色后荧光显微镜下观察凋亡细胞，流式细胞术检测细胞周期、细胞凋亡率及线粒体膜电位（MMP），蛋白质印迹法（Western blot）检测细胞凋亡相关蛋白的表达。结果链球菌蛋白（10～100 mg/L）可显著抑制细胞增殖，使细胞周期阻滞于G2/M期，呈时间和剂量依赖性；链球菌蛋白作用细胞24 h后，MMP显著下降（P＜0．01），凋亡细胞逐渐增多，48 h时各实验组凋亡率明显高于对照组（P＜0．01）；链球菌蛋白（50 mg/L）呈时间依赖性增强细胞P53蛋白表达，抑制B淋巴细胞瘤-2（Bcl-2）蛋白表达，促使细胞色素C（CytC）从线粒体进入胞质，从而激活半胱氨酸天冬氨酸蛋白酶3前体（Procaspase-3）。结论链球菌蛋白可显著抑制人脑多形性胶质母细胞瘤BT325细胞增殖，影响细胞周期分布，通过线粒体途径诱导细胞发生凋亡，由此发挥其抗肿瘤活性作用。
Objective To investigate the mechanisms of streptococcal protein inducing cycle arrest and apoptosis in human cerebral glioblastoma BT325 cell.Methods Human cerebral glioblastoma BT325 cells were cultured in vitro,and methlcyclopentadienyl manganese tricarbonyl （MTT）assay was used to determine the proliferation activity of BT325 cells. The morphological changes of BT325 cells were observed by the phase-contrast microscopy.The apoptotic cells were observed after fluorescence staining.Flow cytometry was used to determine the cell cycle,the rate of cell apoptosis and the change of mitochondrial membrane potential （MMP）.The expression of apoptosis-related proteins was determined by Western blot.Results Streptococcal protein （1 0-1 00 mg/L）inhibited the proliferation of BT325 cells significantly and induced G2/M phase arrest in time- and dose-dependent manners.MMP declined （P 〈0.01 ）,and apoptotic cells gradually increased after BT325 cells were treated with streptococcal protein for 24 h.The rate of cell apoptosis in determination group was significantly higher than that in control group at 48h （P〈0.01 ）.In a time-dependent manner, streptococcal protein （50 mg/L）up-regulated the expression of cell P53 protein,down-regulated the expression of B-cell lymphoma-2（Bcl-2）,led to the release of mitochondrial cytochrome C（CytC）from mitochondria into cytoplasm,and then caused the activation of the precursor of cysteinyl aspartate specific proteinase 3 （Procaspase-3 ）. Conclusions Streptococcal protein may exert its anticancer activity through inhibiting the proliferation of human cerebral glioblastoma BT325 cells,effecting the distribution of cell cycle and inducing cell apoptosis by mitochondrial-dependent pathway.
出处
《检验医学》
CAS
2014年第4期363-368,共6页
Laboratory Medicine
基金
北京市教育委员会科技发展计划项目(KM2004 10025008)
