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目的 研究用眼镜蛇毒因子 (CVF)激活补体对培养内皮细胞 (EC)形态和功能的影响。方法 用M 199培养基培养猪主动脉EC ,了解补体引起EC损伤用台盼蓝染色法 ,通过光镜和电镜观察EC损伤的形态结构 ,用免疫组化显示内皮素 1(ET 1)和碱性成纤维细胞生长因子 (bFGF) ,用发色底物检测法测定组织型纤溶酶原激活物 (t PA)活性。结果 免疫组化染色显示培养细胞胞质中有Ⅷ因子阳性物质 ,确认为EC。在新鲜血清存在下 ,CVF对EC损伤有明显的量效和时效关系 ,作用在 6h达峰值。损伤EC发生变形 ,表现为收缩变圆 ,突起及连接减少 ,胞膜及核膜缺损 ,胞质空泡增多。EC分泌ET 1及bFGF增多 ,而分泌t PA减少。CD5 9有明显的保护作用。结论 CVF激活补体能引起浓度和时间依赖性EC损伤 ,其形态和功能改变有利于凝血 ,可能通过补体攻膜复合物的损伤所致。
Objective To investigate the effects of cobra venom factor (CVF) activation on the morphology and function of cultured endothelial cells (ECs). Methods Porcine aortic ECs were cultured in M 199 medium, EC injury induced by complement was detected by trypan blue staining, and morphological changes of EC were observed by light microscope and electron microscope. Endothelin 1 (ET 1) and alkali Fibroblast growth factor (bFGF) was assayed for tissue tPA activity using a chromogenic substrate assay. Results Immunohistochemical staining showed that there were Ⅷ-factor positive cells in the cytoplasm of cultured cells, which was confirmed as EC. In the presence of fresh serum, CVF had significant dose-effect and time-dependent effects on EC injury, with peak at 6h. Degeneration EC damage, the performance of the contraction round, the protrusions and connections to reduce the membrane and nuclear membrane defects, cytoplasmic vacuolization increased. EC secretion of ET 1 and bFGF increased, while the secretion of t PA decreased. CD5 9 has a significant protective effect. Conclusion CVF activation of complement can cause concentration-dependent and time-dependent EC injury. Its morphological and functional changes favor coagulation, which may be caused by the damage of complement membrane complex.