Caveolae对血管平滑肌细胞胞内钙浓度调节作用的研究

发布时间：2018-05-04 19:13

  本文选题：血管平滑肌细胞 + 小窝　； 参考：《第三军医大学》2014年硕士论文

【摘要】：研究背景和目的： 蛛网膜下腔出血(subarachnoid hemorrhage,SAH)是由于外伤或颅内动脉瘤破裂导致的血液成分进入蛛网膜下腔后所引起的一组临床症状，而脑血管痉挛(cerebralvasospasm，CVS)则是SAH后最严重的并发症，常引起严重脑组织缺血或迟发缺血性脑损害,甚至导致脑梗塞，为患者致残和致死的主要因素。而引起CVS发生的直接原因是SAH后血管平滑肌细胞(vascular smooth muscle cells，VSMCs)上钙通道表达及活性发生变化，引起胞内钙浓度升高，通过激活肌球蛋白轻链激酶（myosin light chainkinase,MLCK）途径引起平滑肌收缩。 小窝(caveolae)是细胞质膜表面特异性的内陷微区，大小约50-100nm形如烧瓶状。它是细胞质膜中相对稳定且流动性不大的区域，被称为细胞内外物质交换和信号传导的平台。小窝蛋白-1(caveolin-1)是caveolae的表面标记功能蛋白，具有脚手架样结构(caveolin scaffolding domain,CSD),聚集了血小板源性生长因子受体、胰岛素受体和血管紧张素受体等多种膜受体，是小窝的发挥生理作用的功能蛋白。至今Caveolin-1/caveolae在调节VSMCs钙通道表达及活性，进而影响CVS发生和发展的报道较少。为此本研究通过改变体外培养VSMCs的caveolin-1蛋白表达，研究caveolin-1表达的改变对VSMCs胞内钙浓度([Ca2+]i)的变化带来的影响，以探讨caveolin-1/caveolae在脑血管痉挛中的可能作用。 方法： 1、分离SD大鼠颈总动脉，利用组织贴块法体外培养血管平滑肌细胞，通过对细胞形态学观察和平滑肌细胞特异性α-肌动蛋白(α-actin)免疫荧光染色进行鉴定。 2、蛋白印迹Western blot法检测1mmol/L、2.5mmol/L和5mmol/L浓度MβCD溶液处理后VSMCs caveolin-1表达水平。 3、蛋白印迹Western blot法检测20ug/L、50ug/L、100ug/L浓度TNF-α溶液孵育后VSMCs caveolin-1表达水平。 4、通过MβCD(5mmol/L)和炎症因子TNF-α(100ug/L)的不同处理将细胞分为四组：A.对照组；B. MβCD组；C. TNF-α组；D. TNF-α+MβCD组。采用TILL荧光钙成像系统检测活细胞在ET-1(100nmol/L)刺激下胞内钙荧光强度(FI)变化情况。 结果： 1、经过对细胞形态学观察和α-actin免疫荧光染色鉴定，原代成功分离和培养大鼠颈总动脉VSMCs。 2、在不同浓度MβCD作用下使细胞caveolin-1蛋白含量随MβCD浓度的升高而降低。caveolin-1蛋白含量5mmol/L MβCD处理组较对照组下降明显(P0.01)。 3、在不同浓度TNF-α作用下使细胞caveolin-1蛋白含量随TNF-α浓度的升高而升高。caveolin-1蛋白含量100ug/L浓度TNF-α处理组较对照组上升明显(P0.01)。 4、TILL荧光钙成像系统检测四组细胞结果显示1)、各组细胞静息状态R0分别为A:0.5916±0.0820,B:0.5753±0.0790, C:0.6551±0.0837,D:0.6105±0.0804,四间细胞组间无显著差异(P0.05)。2)、四组细胞经相同刺激后胞内钙荧光强度(FI)分别较静息状态上升,A组上升(115±17)％；B组上升(67±15)％，，较A组差异显著(P0.05)；C组上升(279±21)％,较A组差异显著(P0.01)；D组上升(88±16)％,较C组差异显著(P0.01)。 结论： 1、5mmol/L MβCD能有效干扰破坏细胞膜caveolae结构，可显著下调VSMCscaveolin-1蛋白表达。 2、100ug/L TNF-α孵育可显著上调VSMCs caveolin-1蛋白表达。 3、caveolin-1蛋白表达上调后可明显增加ET-1诱导的VSMCs胞内钙浓度升高程度，而MβCD有效干扰破坏caveolae结构后导致caveolin-1蛋白表达下调后，可明显降低ET-1诱导的VSMCs胞内钙浓度升高程度，caveolin-1/caveolae可能是抑制脑血管痉挛发生的重要靶点。
[Abstract]:Research background and purpose:
Subarachnoid hemorrhage (subarachnoid hemorrhage, SAH) is a group of clinical symptoms caused by blood components caused by traumatic or intracranial aneurysm rupture into the subarachnoid cavity, and cerebral vasospasm (cerebralvasospasm, CVS) is the most serious complication after SAH, which often causes severe cerebral ischemia or delayed ischemic brain damage. The main cause of disability and death is the cause of cerebral infarction. The direct cause of the occurrence of CVS is the changes in the expression and activity of calcium channels on the vascular smooth muscle cells (vascular smooth muscle cells, VSMCs) after SAH, causing the increase of intracellular calcium concentration, and by activating the myosin light chain kinase (myosin light chainkinase, MLCK). The diameter caused the contraction of the smooth muscle.
Caveolae is a specific internal subsidence microarea on the surface of the cell membrane. It is about 50-100nm shaped like a flask shape. It is a relatively stable and fluidity area in the plasma membrane of the cell. It is called a platform for the exchange of substances and signals inside and outside cells. The fossa protein -1 (caveolin-1) is a surface labeled functional protein of caveolae and has a scaffolding sample. Caveolin scaffolding domain (CSD), which congregates a variety of membrane receptors, such as platelet derived growth factor receptor, insulin receptor and angiotensin receptor, is a functional protein that plays a physiological role in the fossa. So far, Caveolin-1/caveolae has been less reported in regulating the expression and activity of VSMCs calcium channels and affecting the occurrence and development of CVS. In this study, the effect of the change of caveolin-1 expression on the intracellular calcium concentration ([Ca2+]i) in VSMCs was investigated by changing the expression of caveolin-1 protein in VSMCs in vitro, so as to explore the possible role of caveolin-1/caveolae in cerebral vasospasm.
Method锛

本文编号：1844288