肿瘤坏死因子诱导内皮细胞Ca^(2+)时空变化的激光共聚焦显微测定

THE DETERMINATION OF THE SPATIAL AND DYNAMIC CHANGES OFCa^(2+)INDUCED WITH TUMOR NECORSIS FACTOR IN SINGLE ENDOTHELIAL CELL BY LASER SCANNING CONFOCAL MICROSCOPY

目的 研究肿瘤坏死因子 (TNFα)对培养的单个内皮细胞内游离Ca2 +浓度 [(Ca2 )i]的影响 ,以探讨TNFα介导休克的细胞机制。方法 人脐静脉内皮细胞株 (ECV30 4 )接种于 35mm含有 2mlDMEM培养基的组织培养盘中培养。TNFα诱导的单个内皮细胞 [Ca2 +]i时空变化由激光扫描共聚焦显微系统 (LSCM)和Fluo - 3/AM荧光探剂标记技术测定。结果 TNFα使单个内皮细胞 [Ca2 +]i呈剂量依赖性升高 ,在 60s内达到峰值 ,然后下降并保持在基础水平之上。共聚焦扫描图像显示细胞核区 [Ca2 +]i升高比胞浆区明显 ,下降比胞浆区慢。结论 TNFα显著诱导内皮细胞 [Ca2 +]i升高 ,可能是TNFα介导休克和组织损伤的重要机制之一。

Objective To study the effects of tumor necrosis factor (TNF α) on intracellular free Ca 2+ concentration ([Ca 2+ ]i) in single cultured endothelial cell, and to explore the mechanisms of TNF α-mediated shock. Methods Human umbilical vein endothelial cell strains(ECV304) was seed in 35-mm tissue culture dish with 2 ml DMEM culture medium. The spatial distribution and the dynamic changes of [Ca 2+ ]i induced by TNF α in single endothelial cell were determined using laser scanning confocal microscopy ( LSCM ) and the highly fluorescent Ca 2+ -sensitive indicator Fluo-3/AM. Results [Ca 2+ ]i in single endothelial cell after stimulation of TNF α rapidly increased in a dose-dependent manner and arrived at the peak value within 60 seconds, afterwards, decreased and kept above the basal level. The confocal scanning image showed that [Ca 2+ ]i elevation was more obvious in nuclear than in cytoplasma, and decreased slowly.Conclusion TNF α markedly induces elevation of [Ca 2+ ]i in single endothelial cell, it may be an important mechanism of TNF α-induced shock and tissue injury.