初探内皮细胞膜微粒（EMPs）于体外对内皮细胞功能及凋亡的影响

【作者】 郑敏哲；

【导师】 翁习生；

【作者基本信息】 中国协和医科大学 ， 临床医学， 2008， 博士

【摘要】 研究背景:股骨头微循环障碍、血液高凝倾向和小血管阻塞是激素性股骨头坏死诸多发病机制中比较重要的机制。内皮细胞膜微粒(Endothelial Microparticles,EMPs)是由内皮细胞分泌,磷脂膜包被的微小囊泡结构,具有影响血液凝血系统、血管舒张功能,且与多种内皮损伤性血栓及炎症疾病相关。同时EMPs参与细胞凋亡、活化、炎症细胞募集等多种病生理过程。研究目的:探讨体外试验中,激素对于内皮细胞EMPs释放的影响.探讨EMPs对内皮细胞自身的影响,包括内皮功能和凋亡两方面,进而初探EMPs在激素相关性股骨头缺血坏死机制中的作用.研究方法:实验对象:实验中选择体外培养的人脐静脉内皮细胞株(EAhy926)。实验刺激物:激素对内皮细胞实验中使用DEX(dexamethasone)激素,EMPs对内皮细胞影响实验则选择内皮细胞自分泌的EMPs。检测方法:EMPs和内皮细胞共培养一段时间后倒置相差显微镜观察细胞形态学变化,用流式细胞仪检测EMPs数量,随后用Bradford法测BSA(BovineSerum Albumin)浓度值,判断内皮细胞通透性变化。以Caspase-3试剂盒和Annexin-V FITC试剂盒测定细胞凋亡状况,RT-PCR检测内皮细胞凋亡基因的表达。研究结果:1.流式细胞仪分析:10mM浓度激素刺激24h,内皮细胞释放EMPs达到最大。2.中、高浓度EMPs对内皮细胞通透性产生损伤,低浓度无明显差异。3.试剂盒检测结果:高中低浓度EMPs内皮细胞Caspase-3、PS(Phosphatidylserine)活性表达均高于对照。4.Realtime-PCR、RT-PCR分析凋亡基因:高、中浓度EMPs组APAF-1、AIF基因表达较对照增高,低浓度组没有明显差异。结论:1.DEX在10mM,24h条件下刺激内皮细胞产生最大量的EMPs2.7.66*10~4和7.66*10~3个/ml浓度EMPs对内皮细胞通透性可引起损伤,但7.66~*10~2个/mlEMPs则不会对内皮产生明显损伤。3.7.66*10~4、7.66*10~3、7.66*10~2个/ml浓度EMPs均促内皮细胞凋亡发生;Caspase-3、PS活性表达均增高。4.7.66*10~*和7.66*10~3个/ml浓度EMPs可使内皮细胞APAF-1、AIF基因表达增高,7.66*10~2个/ml浓度EMPs则没有明显作用。更多还原

【Abstract】 Background:microcirculation disorder,hypercoagulation state and microvascular occlusion are important pathways in the pathogenesis of Avascular Necrosis of Femoral Head(ANFH).Endothelial microparticles(EMPs) are phospholipid vesicles derived from endothelial cells.It has been reported that EMPs can influence plasma coagulation system and vasodilatation.Also,they are implicated in inflammation and vascular thrombosis associated with endothelial injury.Moreover,it has been shown that EMPs are involved in cell signaling,apoptosis and recruitment of inflammatory cells.Object:To investigate the effect of glucocortoid on EMPs releasing in vitro. To investigate the effect of EMPs on HUVECs,including endothelial function and apoptosis and to study the contribution of EMPs on ANFH.Material and methods:Object:Human Umbilical Vein Endothelial Cells(HUVECs) in vitro(EAhy926).Stimulator:dexamethasone(DEX) and EMPs released by HUVECs.Method:After cultured with EMps,HUVECs were examined under microscope, and then Mps were counted using flowcytometry.The value of bovine serum albumin was determined by Bradford method to evaluate the permeability of HUVECs. Cellular apoptosis were examined using Caspase-3 and Annexin-V FITC kit.The expression of genes associated with apoptosis were detected by reverses transcription-PCR.Results:Flowcytometry analysis showed that EMPs were released at maximum concentration after 24 hours stimulated by 10mM DEX.Moderate and high concentrations of EMPs caused injury to the permeability of HUVECs,while low concentration of EMP didn’t.All concentrations of EMPs induced higher activity of Caspase-3 and PS than control did.Moderate and high concentration groups showed increased expression of APAF-1,AIF gene in comparison with control,while low concentration group didn’t.Conclusion:DEX(10mM,24h) stimulated HUVECs to release EMPs at maximum concentration.EMPs at the concentration of 7.66*104 and 7.66*103/ml caused injury to the permeability of HUVECs,while 7.66*102/ml did not show significant difference from control.All three concentrations of EMPs induced HUVECs apoptosis with increased activity of Caspase-3 and PS.EMPs at the concentration of 7.66*104 and 7.66*103/ml showed increased expression of APAF-1,AIF gene in comparison with control,while low concentration group didn’t.更多还原