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

发布时间：2018-01-03 03:16

本文关键词：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　出处：《中国协和医科大学》2008年博士论文　论文类型：学位论文

【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EMPs 2.7.66*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。
[Abstract]:Background: Femoral head microcirculation disorder. Hypercoagulability and small vessel occlusion are the most important mechanisms in the pathogenesis of steroid-induced femoral head necrosis. Endothelial Microparticles. EMPs are tiny vesicles secreted by endothelial cells and coated with phospholipid membrane, which affect the blood coagulation system and vasodilation function. EMPs is involved in many physiological processes such as apoptosis, activation and recruitment of inflammatory cells. Objectives of the study: To investigate the effect of hormone on the release of EMPs from endothelial cells in vitro. To explore the effect of EMPs on endothelial cell itself, including endothelial function and apoptosis, and to explore the role of EMPs in the mechanism of steroid-related avascular necrosis of femoral head. Research methods: Participants: human umbilical vein endothelial cells (HUVECs) were cultured in vitro. Experimental irritants:. The effect of EMPs on endothelial cells was determined by using DEXDX dexamethasoneone. The autocrine EMPs of endothelial cells were selected. Methods the morphologic changes of EMPs were observed by inverted phase contrast microscope after co-cultured with endothelial cells for a period of time. Flow cytometry was used to detect the number of EMPs. Then the concentration of BSA(BovineSerum was measured by Bradford method. The changes of endothelial cell permeability were evaluated. Apoptosis was measured by Caspase-3 kit and Annexin-V FITC kit. The expression of apoptotic gene in endothelial cells was detected by RT-PCR. Results of the study: 1. Flow cytometry analysis showed that the release of EMPs from endothelial cells reached the highest level after 24 h stimulation with 10 mm concentration of hormone. 2. The permeability of endothelial cells was damaged by high concentration of EMPs, but there was no significant difference in low concentration. 3. The expression of Caspase-3 Phosphatidylserine (Phosphatidyl serine) activity in EMPs endothelial cells was significantly higher than that in the control group. 4.Realtime-PCR- RT-PCR analysis of apoptotic genes: APAF-1AIF gene expression was higher in high and medium concentration EMPs group than that in control group. There was no significant difference in the low concentration group. Conclusion: 1. DEX stimulated endothelial cells to produce the largest amount of EMPs at 10 mm ~ (-1) for 24 h. 2.7.66 ~ 10 ~ 3 / ml EMPs and 7.66 ~ 10 ~ 3 / ml EMPs could damage the permeability of endothelial cells. However, 7.66% 10 ~ 2 ml EMPs did not significantly damage the endothelium. 3.7.66 ~ 10 ~ 2 / ml EMPs promoted the apoptosis of endothelial cells. The activity of Caspase-3 and PS were all increased. 4.7.66 ~ 10 ~ 3 / ml EMPs increased the expression of APAF-1 / AIF gene in endothelial cells. There was no obvious effect of EMPs at the concentration of 7.66 ~ 10 ~ 2 / ml.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R363

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