曲霉菌素诱导人胚肾细胞毒性机制(英文)

Mechanism of cytotoxicity of human embryonic kidney cells induced by gliotoxin

目的 研究曲霉菌素诱导人胚肾细胞毒性作用机制。方法 结晶甲紫法用于细胞存活率研究。琼脂糖凝胶及Burton法研究细胞核DNA断裂片段。以水解特异性底物Ac DEVD AMC活性为研究指标 ,测定胞浆半胱天冬酶 (caspase) 3类蛋白酶活性。采用蛋白质印迹法检测细胞半胱天冬酶 3蛋白表达。采用荧光标记探针、流式细胞仪技术研究细胞核DNA核型及细胞活性氧的产生。结果 曲霉菌素浓度依赖性地诱导人胚肾细胞凋亡 ,最大效应浓度为 1 .0mg·L-1 。BAF ,半胱天冬酶 3蛋白抑制剂和N 乙酰半胱氨酸 (活性氧抑制剂 )能显著性抑制曲霉菌素诱导人胚肾细胞凋亡作用。结论 半胱天冬酶类及活性氧调节曲霉菌素诱导的人胚肾细胞凋亡。

AIM To study the mechanism underlying gliotoxin-induced c yt otoxicity of human embryonic kidney (HEK) cells. METHODS Crystal vi olet assay was used to determine cell viability. DNA fragmentation of HEK cells was measured based on Burton′s method. The activity of caspase-3-like protea se s was measured as increases in hydrolysis of fluorogenic tetrapeptide substrate, Ac-DEVD-7-amino-4-methylcoumarin and caspase-3 protein abundance was obse rved by Western blot. Based on fluorescence probe label method, DNA content and reactive oxygen species (ROS) of HEK cells were detected by flow cytometry. RESULTS Gliotoxin induced HEK cell death in a concentration-dependent manner within 0.4-1.0 mg·L -1 . Under gliotoxin treatment at 1.0 mg·L -1 , cell membrane of HEK cells k e pt intact associated with hypodiploid nuclei and DNA fragmentation which suggest ed gliotoxin killed HEK cells viaapoptosis. Boc-aspartyl (OMe)-fl uoromethylketone (BAF) and z-DEVD.fmk, commonly used as caspase-3-like pro teases inhibitor, significantly abolished gliotoxin-induced cell death at 100 and 200 μmol·L -1 , respectively, suggesting the cytotoxicity induced by g l iotoxin was mediated by caspases. N-acetylcysteine concentration-d ependently attenuated the HEK cells death induced by gliotoxin, significantly in hibited the generation of ROS of HEK cells upon exposure with gliotoxin, which i ndicated that ROS was involved in the cytotoxicity of HEK cells induced by gliot oxin. CONCLUSION Gliotoxin-induced cytotoxicity of HEK cells pro ceeded viaapoptosis, which was mediated by caspases and ROS.