【摘要】 目的:研究肿瘤坏死因子（TNFα）对单个内皮细胞胞内游离Ca²⁺浓度([Ca²⁺]_i)的影响及维拉帕米（Ver）、噻庚啶（Cyp）和山莨菪碱（Ani）对TNFα诱导[Ca²⁺]_i变化的影响,以探讨TNFα介导休克和Cyp、Ani的抗休克的机制。方法:人脐静脉内皮细胞株（ECV304）接种于35mm含有2mL DMEM培养基的组织培养盘中培养。Fluo-3/AM负载细胞,激光扫描共聚焦显微技术测定单个内皮细胞[Ca²⁺]_i。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。维拉帕米1和2,噻庚啶30和60或山莨菪碱20和40μmol/L均能显著抑制由TNFα 1.2nmol/L诱导的单个内皮细胞[Ca²⁺]_i升高。结论:TNFα显著诱导内皮细胞[Ca²⁺]_i升高,可能是TNFα介导休克的重要机制;维拉帕米、噻庚啶和山莨菪碱对TNFα诱导的[Ca²⁺]_i升高有拮抗作用,可能是噻庚啶和山莨菪碱抗休克作用的机制之一。更多还原

【Abstract】 AIM: To study the effect of tumor necrosis factor alpha (TNFa ) on intracellular free Ca2+ concentration ([Ca2+ ]i) and the effects of verapamil (Ver), cyproheptadine (Cyp), and anisodamine (Ani) on TNFa-induced [Ca2+ ]i changes in single endothlial cell, and to explore the mechanisms of TNFa-mediated shock and antishock actions of Cyp and Ani. METHODS: Human umbilical vein endothlial cell strains (ECV304) were seeded in 35-mm tissue culture dish with 2 mL DMEM culture medium. The cultured cells were loaded by Fluo-3/AM. The spatial distribution and the dynamic changes of [ Ca2+ ]; in single endothelial cell were determined by laser scanning confocal microscopy. RESULTS: After stimulation with TNFa, [Ca2+ ]i in single endothelial cell rapidly increased in a concentration-dependent manner and arrived at the peak value within 60 s, afterwards, decreased and kept above the basal level. The confocal scanning image showed that [ Ca2+ ]i elevation was more obvious in nuclear than in cytoplasma and decreased slowly. Ver (1,2 μmol/L), Cyp (30, 60 μmol/L), and Ani (20, 40 μmol/L) markedly inhibited TNFa 1.2 nmol/L-induced [Ca2+ ]i elevation. CONCLUSION: TNFa markedly induces elevation of [Ca2+ ]i in a single endothelial cell, it may be an important mechanism of TNFa-induced shock and tissue injury. That Cyp and Ani obviously suppress TNFa-induced [ Ca2+ ]i elevation probably is one of themechanisms of their antishock effects.更多还原