Thapsigargin对人晶状体上皮细胞系SRA01/04增殖抑制的作用机制  

Effect and Mechanism of Thapsigargin on Proliferation of Human Lens Epithelilal Cells Line SRA01/04

作　　者：姬红培[1,2] 吴明星[1] 彭瑞萍[1] 

机构地区：[1]眼科学国家重点实验室//中山大学中山眼科中心 [2]贵州省人民医院,贵州贵阳550002

出　　处：《中山大学学报（医学科学版）》2007年第6期622-626,共5页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(30572003;39870803);广东省自然科学基金(031724);广州市科技计划项目(2003Z3-E0031)

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72h,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(17/19ku)的表达及阳性细胞表达率。【结果】MTT法测得TG为0.325～5μmol/L时,细胞增殖抑制率变化显著,IC50大约为0.8μmol/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0,0.1,0.5,1,10μmol/L分别处理SRA01/04细胞72h,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10μmol/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。[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- dimethyhhiazol-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 dUTPnick 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 uhrastructure 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 caspase3 was activated by TG,the cleaved caspase3 was expressed in a concentration dependent manner. The positive rate was about 100% at the concentration of 10 μmol/L. [Conclusion] The growth of SRA01/04 cells were inhibited by thapisigargin in a concentration dependent manner in vitro by inducing up-regulation of caspase 3 activation and starting apoptosis pathway. Thapisigargin may be developed as a potential therapeutic drug for posterior capsule opacification prevention.

关 键 词：Thapsigarigin 晶状体上皮细胞 细胞凋亡 CASPASE3 

分 类 号：R770.67[医药卫生—眼科]