Staurosporine诱导小鼠心肌细胞凋亡模型的构建

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 Staurospor

Molecular identification of mouse cardiomyocytes apoptosis induced by staurosporine

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 cardiomyocyte apoptosis induced by staurosporine. METHODS: Neonatal mouse cardiomyocytes were cultured and treated with or without staurosporine. Cellular morphological characteristics, DNA fragmentations, caspase-3 activity, cell viability and cell membrane integrity were observed. RESULTS: Remarkable morphological characteristics of cardiomyocytes apoptosis, including cell shrinkage, condensed chromatin and DNA fragmentations (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 which was time and concentration(1～16 μmol/L) dependent. Myocyte lactate dehydrogenase(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 irreversible tripeptide inhibitor of caspases ZVAD-fmk. CONCLUSION: These data suggest that staurosporine can rapidly and efficiently induce apoptotic death of mouse cardiomyocytes. Caspase-3 may play a critical role in the process of staurosporine induced cardiomyocyte apoptosis.