Staurosporine诱导小鼠心肌细胞凋亡模型的构建(英文)  

Molecular identification of mouse cardiomyocytes apoptosis induced by staurosporine

作　　者：王晓明[1] 臧益民[2] 高峰[2] 龚卫琴[1] 李源[1] 

机构地区：[1]第四军医大学西京医院老年病1科,陕西西安710032 [2]第四军医大学西京医院生理学教研室,陕西西安710032

出　　处：《心脏杂志》2004年第1期4-10,共7页Chinese Heart Journal

摘　　要：目的 :Staurosporine可诱导不同细胞类型的细胞凋亡 ,但尚缺乏其对小鼠心肌细胞作用影响的报道。本研究旨在观察 Staurosporine是否也可引起小鼠心肌细胞的凋亡 ,并对其模型进行分子水平的鉴定。方法 :用 Stau-rosporine处理原代培养的小鼠心肌细胞的直接刺激后 ,观察心肌细胞的形态学变化、DNA片段、半胱天冬蛋白酶(caspase) -3活性、细胞存活率以及细胞膜完整性。结果 :1Staurosporine处理过的心肌细胞具有明显的凋亡形态学特征 :细胞的皱缩、核的浓缩及 DNA梯改变。2 Staurosporine4μmol/ L 对心肌细胞作用 8h后 ,与正常对照组比较细胞存活率降至 3 1.2 % (与对照组比 ,P<0 .0 1) ,且细胞存活率与 Staurosporine的作用浓度和作用时间呈依赖关系 ;同时测定细胞培养液中能反映细胞膜完整性的胞浆内容物乳酸脱氢酶 (L DH)水平 ,发现在 Staurosporine作用 1～ 8h的各个时间点 ,L DH的漏出水平最高未超过 10 % (与对照组相比 ,P>0 .0 5) ;这种高的细胞死亡率与低的细胞膜的损伤率 ,恰恰反映了细胞死亡的性质是凋亡性死亡。3 Staurosporine能激活心肌细胞内的 Caspese-3的活性 ,当使用广谱的 Caspese抑制剂 ZVAD-fmk预处理培养的小鼠心肌细胞后 ,能够有效的阻止上述细胞凋亡的发生 ,从而避免了AIM:Staurosporine has been shown to induce apoptosis in some types of cell. The present study was designed to establish and identify a model of mouse cardio myocyte apoptosis induced by staurosporine. METHODS: Neonatal mouse c ardiomyocytes were cultured and treated with or without staurosporine. Cellular morphological characteristics, DNA fragmentations, caspase-3 activity, cell viability and cell membrane integ r ity were observed. RESULTS: Remarkable morphological charac teristics of cardiomyocy tes apoptosis, including cell shrinkage, condensed chromatin and DNA fragm entations ( DNA-laddering) were observed. After 8 h exposure to 4 μmol/L staurosporine,the cell viability of cardiomyocytes was 31.2%(P<0.01,vs control group) and whi ch was time and concentration(1~16 μmol/L) dependent. Myocyte lactate dehydroge nase(LDH) leakage were changed insignificantly in staurosporine treated cells(1 to 8 h) as compared with the control. In addition, staurosporine induced myocyte apoptosis was inhibited by irrev ersible tripeptide inhibitor of caspases ZVAD-fmk. CONCLUSION : These data sugges t that staurosporine can rapidly and efficiently induce apoptotic death of mouse cardiomyocytes. Caspase-3 may play a critical role in the proc ess of staurosporine induced cardiomyocyte apoptosis.

关 键 词：心肌细胞 小鼠 细胞凋亡 STAUROSPORINE CASPASE-3 乳酸脱氢酶 

分 类 号：Q255[生物学—细胞生物学] R363.13[医药卫生—病理学]