伞枝犁头霉在体外诱导人血管内皮细胞凋亡

目的 研究伞枝犁头霉对人脐静脉血管内皮细胞活性的影响及其机制.方法 采用锥虫蓝染色法分析不同时间段伞枝犁头霉对人血管内皮细胞存活率的影响.细胞凋亡检测试剂盒在荧光显微镜下分析伞枝犁头霉诱导人血管内皮细胞的凋亡情况.流式细胞仪定量检测伞枝犁头霉诱导人血管内皮细胞在不同时间段的凋亡情况,并观察caspase-3抑制剂对凋亡反应的影响.用Western blot法检测伞枝犁头霉引发人血管内皮细胞caspase-3在不同时间段活化情况.结果 锥虫蓝染色法显示伞枝犁头霉以时间依赖的方式抑制人血管内皮细胞的存活率.伞枝犁头霉在共培养第12小时开始抑制人血管内皮细胞存活率(P=0.001).荧光显微镜显示伞枝犁头霉诱导人血管内皮细胞发生凋亡,而并非坏死.流式细胞仪分析显示伞枝犁头霉以时间依赖的方式诱导人血管内皮细胞发生凋亡反应.凋亡细胞在共培养第12小时显著增高(P=0.0036).Caspase-3抑制剂几乎可以完全逆转伞枝犁头霉诱导的凋亡反应.Western blot法显示伞枝犁头霉引起人血管内皮细胞caspase-3活化随时间逐渐增强.结论 伞枝犁头霉在体外试验中可以诱导人血管内皮细胞发生凋亡.该凋亡信号的传导是通过caspase级联反应.

Absidia corymbifera induces apoptosis of human umbilical vein endothelial cells in vitro

Objective To analyze the influence of Absidia corymbifera on cell activity of human umbilical vein endothelial cells (HUVEC) as well as the related mechanism. Methods Time course analy sis of the influence of A. corymbifera on cell viability of HUVEC was determined by cell counting after Trypan blue staining. Apoptosis of HUVEC induced by A. corymbifera was observed under fluorescence microscope after treatment with apoptosis detection kit. Time course analysis of HUVEC apoptosis induced by A. corymbifera was detected by flow cytometry quantitatively. Effect of caspase-3 inhibitor on A. corymbifera associated apoptosis was also evaluated at the same time. Activation of caspase-3 inside HUVEC was detected by Western blot. Results A. corymbifera inhibited cell viability of HUVEC in a time-dependent manner by Trypan blue staining. After 12 hours' co-culture, A. corymbifera began to show suppression on cell viability (P =0. 001 ). Fluorescence microscope observation revealed A. corymbifera induced apoptosis of HUVEC instead of necrosis. Flow cytometry analysis showed A. corymbifera induced apoptosis of HUVEC in a time-dependent manner. A. corymbifera began to show obvious effect on apoptosis after 12 h co-culture (P =0.0036). Moreover, A. corymbifera-associated apoptosis was almost abrogated completely by caspase-3 inhibitor. Western blot analysis demonstrated that A. corymbifera triggered the activation of caspase-3 inside HUVEC in a timedependent fashion. Conclusion A. corymbifera induces apoptosis of HUVEC in vitro. Such apoptotic signal is transmitted through caspase cascade reaction.