摘要
目的:比较肿瘤坏死因子α(TNF-α)和内毒素脂多糖(LPS)对人脑微血管内皮细胞(HBMVECs)凝血和纤溶活性的影响。方法:从人脑组织分离、纯化和培养HBMVECs,通过细胞免疫荧光染色、流式细胞术和电子显微镜对HBMVECs进行鉴定。检测TNF-α和LPS刺激前后HBMVECs凝血纤溶活性变化:一期凝固法检测凝血活性(PCA);酶联免疫吸附试验(ELISA)和实时荧光定量聚合酶链反应检测细胞组织因子(TF)、血栓调节蛋白(TM)、组织纤溶酶原激活剂(t-PA)、纤溶酶原激活物抑制剂-1(PAI-1)的水平。结果:分离、纯化的HBMVECs表达内皮细胞典型的标志分子vWF、CD31,并具有摄取乙酰化低密度脂蛋白的能力。LPS和TNF-α促进HBMVECs的凝血活性,上调PCA和TF的产生,抑制抗凝血活性,下调TM的表达;并明显促进纤溶活性t-PA和PAI-1的表达。通过比较PCA/TM发现,TNF-α对HBMVECs凝血活性的影响明显强于LPS。结论:TNF-α在感染性中枢神经系统疾病中可能是导致脑微血管内皮细胞功能障碍的主要因素之一。
Objective：To compare the effects of tumor necrosis factor-α （TNF-α）and E.coli lippolysaccharide （LPS）on the coagulation/fibrinolytie activity of human brain mierovascular endothelial cells（HBMVECs）in vitro. Methods：HBMVECs were isolated from human brain tissue and purified by UEA-1 conjungated magnetic beads.HBMVECs were identified by immunofluoreseence staining,flow cytometry （FCM） and electron microscopy.The eoagulation/fibrinolytic activities of HBMVECs were investigated after stimulated by TNF-α and LPS.The procoagulant activity （PCA）was assessed by way of one-stage recalcification clotting time assay.The expressions of tissue factor（TF）,thrombomodulin（TM）,tissue-type plasminogen activator（t-PA）and plasminogen activator-inhibitor-1 （PAI-1）were detected by real-time PCR and ELISA.Results：The cultured HBMVECs expressed classic endothelial markers CD31,von willebrand factor and uptake high level of Dil-Ac-LDL.In response to TNF-α or LPS,HBMVECs had decreased level of TM and increased level of PCA,TF,t-PA and PAI-1,but the effect of TNF-α was significantly stronger than LPS.Conclusion：TNF-α might influence the activities of coagulation and fibrinolysis in HBMVECs,and results in dysfunction of HBMVECs,whieh might play a critical role in immunopathology of infectious central nervous system diseases.
出处
《中日友好医院学报》
2009年第5期275-279,F0002,共6页
Journal of China-Japan Friendship Hospital
关键词
脑
内皮细胞
肿瘤坏死因子
内毒素脂多糖
凝血纤溶活性
brain
endothelial cell
tumor necrosis factor
E.coli lippolysaccharide
coagulation fibrinolytic activity
