本文關(guān)鍵詞：初探內(nèi)皮細胞膜微粒（EMPs）于體外對內(nèi)皮細胞功能及凋亡的影響　出處：《中國協(xié)和醫(yī)科大學》2008年博士論文　論文類型：學位論文

  更多相關(guān)文章： 股骨頭壞死 細胞膜微粒 內(nèi)皮細胞 凋亡

【摘要】： 研究背景: 股骨頭微循環(huán)障礙、血液高凝傾向和小血管阻塞是激素性股骨頭壞死諸多發(fā)病機制中比較重要的機制。內(nèi)皮細胞膜微粒(Endothelial Microparticles,EMPs)是由內(nèi)皮細胞分泌,磷脂膜包被的微小囊泡結(jié)構(gòu),具有影響血液凝血系統(tǒng)、血管舒張功能,且與多種內(nèi)皮損傷性血栓及炎癥疾病相關(guān)。同時EMPs參與細胞凋亡、活化、炎癥細胞募集等多種病生理過程。 研究目的: 探討體外試驗中,激素對于內(nèi)皮細胞EMPs釋放的影響. 探討EMPs對內(nèi)皮細胞自身的影響,包括內(nèi)皮功能和凋亡兩方面,進而初探EMPs在激素相關(guān)性股骨頭缺血壞死機制中的作用. 研究方法: 實驗對象:實驗中選擇體外培養(yǎng)的人臍靜脈內(nèi)皮細胞株(EAhy926)。 實驗刺激物:激素對內(nèi)皮細胞實驗中使用DEX(dexamethasone)激素,EMPs對內(nèi)皮細胞影響實驗則選擇內(nèi)皮細胞自分泌的EMPs。 檢測方法:EMPs和內(nèi)皮細胞共培養(yǎng)一段時間后倒置相差顯微鏡觀察細胞形態(tài)學變化,用流式細胞儀檢測EMPs數(shù)量,隨后用Bradford法測BSA(BovineSerum Albumin)濃度值,判斷內(nèi)皮細胞通透性變化。以Caspase-3試劑盒和Annexin-V FITC試劑盒測定細胞凋亡狀況,RT-PCR檢測內(nèi)皮細胞凋亡基因的表達。 研究結(jié)果: 1.流式細胞儀分析:10mM濃度激素刺激24h,內(nèi)皮細胞釋放EMPs達到最大。 2.中、高濃度EMPs對內(nèi)皮細胞通透性產(chǎn)生損傷,低濃度無明顯差異。 3.試劑盒檢測結(jié)果:高中低濃度EMPs內(nèi)皮細胞Caspase-3、PS(Phosphatidylserine)活性表達均高于對照。 4.Realtime-PCR、RT-PCR分析凋亡基因:高、中濃度EMPs組APAF-1、AIF基因表達較對照增高,低濃度組沒有明顯差異。 結(jié)論: 1.DEX在10mM,24h條件下刺激內(nèi)皮細胞產(chǎn)生最大量的EMPs 2.7.66*10~4和7.66*10~3個/ml濃度EMPs對內(nèi)皮細胞通透性可引起損傷,但7.66~*10~2個/mlEMPs則不會對內(nèi)皮產(chǎn)生明顯損傷。 3.7.66*10~4、7.66*10~3、7.66*10~2個/ml濃度EMPs均促內(nèi)皮細胞凋亡發(fā)生;Caspase-3、PS活性表達均增高。 4.7.66*10~*和7.66*10~3個/ml濃度EMPs可使內(nèi)皮細胞APAF-1、AIF基因表達增高,7.66*10~2個/ml濃度EMPs則沒有明顯作用。
[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.
【學位授予單位】：中國協(xié)和醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R363

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相關(guān)期刊論文 前1條

1 孫偉;;股骨頭壞死的病因、病理和發(fā)病機制[J];中華全科醫(yī)師雜志;2006年02期

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本文編號：1372133