Thapsigargin对人晶状体上皮细胞系SRA01-04增殖抑制作用机制.docVIP

Thapsigargin对人晶状体上皮细胞系SRA01/04增殖抑制作用机制 　作者：姬红培， 吴明星， 彭瑞萍 【摘要】 【目的】 研究thapsigargin（TG）对体外培养的人晶状体上皮细胞系SRA01/04增殖的影响，并探讨其分子机制。【方法】 不同浓度TG处理SRA01/04细胞72 h，噻唑蓝比色法（MTT）检测细胞增殖；流式细胞仪（PI法）检测细胞周期改变；透射电镜观察细胞超微结构变化；TUNEL法检测细胞核中DNA的断裂情况；激光共焦显微镜和流式细胞仪检测Caspase3活性亚单位（17/19 ku）的表达及阳性细胞表达率。【结果】 MTT法测得TG为0.325～5 μmol/L时，细胞增殖抑制率变化显著，IC50大约为0.8 μmol/L;透射电镜显示TG处理SRA01/04细胞膜皱缩，染色体浓缩、边集、核膜裂解、凋亡小体形成;流式细胞仪（PI法）及TUNEL法检测TG浓度为0，0.1，0.5，1，10 μmol/L分别处理SRA01/04细胞72 h，细胞凋亡率随浓度的增加而增加，呈浓度依赖性;TG处理后细胞Caspase3阳性表达率也呈浓度依赖性增加,TG为10 μmol/L时，几乎每个细胞浆内Caspase3都被激活。【结论】 Thapsigargin可能通过激活Caspase3为诱导体外培养的人晶状体上皮细胞系SRA01/04细胞凋亡而抑制其增殖，TG为预防后发性白内障的药物研究提供了基础。 【关键词】 Thapsigarigin； 晶状体上皮细胞； 细胞凋亡； Caspase3 Effect and Mechanism of Thapsigargin on Proliferation of Human Lens Abstract: 【Objective】 To investigate the effect of thapsigargin (TG) on the proliferation of human lens epithelial cells line SRA01/04 in vitro and underlay the molecular mechanism. 【Methods】 SRA01/04 cells were exposed to TG for 72 h, the drug-free medium was as control. Effect of TG on cell growth was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Ultrastructure was observed by transmission electron microscopy. DNA fragmentation was measured by terminal deoxynucleotidyl transferase-mediated dUTP-nick end-labeling (TUNEL). Cell cycle analysis was determined by flow cytometry (FCM) with propidum iodine (PI) staining. Expression of cleaved caspase 3 protein was detected by confocal microscopy and flow cytometry. 【Results】 The growth of SRA01/04 cells were inhibited obviously by TG at concentration ranged from 0.325 to 5 μmol/L. The ultrastructure of treated SRA01/04 cells showed typically apoptotic characteristics, including chromatin condensation, chromatin crescent formation, nucleus fragmentation and apoptotic body formation. Exposed to 0, 0.1，0.5，1.0, 10 μmol/L TG for 72 h, the apoptotic rate by flow cytometry (PI means) and TUNEL were increased in a concentration-dependent manner. The expression of ca