本文選題：血管平滑肌細(xì)胞 + 小窩　； 參考：《第三軍醫(yī)大學(xué)》2014年碩士論文

【摘要】：研究背景和目的： 蛛網(wǎng)膜下腔出血(subarachnoid hemorrhage,SAH)是由于外傷或顱內(nèi)動(dòng)脈瘤破裂導(dǎo)致的血液成分進(jìn)入蛛網(wǎng)膜下腔后所引起的一組臨床癥狀，而腦血管痙攣(cerebralvasospasm，CVS)則是SAH后最嚴(yán)重的并發(fā)癥，常引起嚴(yán)重腦組織缺血或遲發(fā)缺血性腦損害,甚至導(dǎo)致腦梗塞，為患者致殘和致死的主要因素。而引起CVS發(fā)生的直接原因是SAH后血管平滑肌細(xì)胞(vascular smooth muscle cells，VSMCs)上鈣通道表達(dá)及活性發(fā)生變化，引起胞內(nèi)鈣濃度升高，通過(guò)激活肌球蛋白輕鏈激酶（myosin light chainkinase,MLCK）途徑引起平滑肌收縮。 小窩(caveolae)是細(xì)胞質(zhì)膜表面特異性的內(nèi)陷微區(qū)，大小約50-100nm形如燒瓶狀。它是細(xì)胞質(zhì)膜中相對(duì)穩(wěn)定且流動(dòng)性不大的區(qū)域，被稱為細(xì)胞內(nèi)外物質(zhì)交換和信號(hào)傳導(dǎo)的平臺(tái)。小窩蛋白-1(caveolin-1)是caveolae的表面標(biāo)記功能蛋白，具有腳手架樣結(jié)構(gòu)(caveolin scaffolding domain,CSD),聚集了血小板源性生長(zhǎng)因子受體、胰島素受體和血管緊張素受體等多種膜受體，是小窩的發(fā)揮生理作用的功能蛋白。至今Caveolin-1/caveolae在調(diào)節(jié)VSMCs鈣通道表達(dá)及活性，進(jìn)而影響CVS發(fā)生和發(fā)展的報(bào)道較少。為此本研究通過(guò)改變體外培養(yǎng)VSMCs的caveolin-1蛋白表達(dá)，研究caveolin-1表達(dá)的改變對(duì)VSMCs胞內(nèi)鈣濃度([Ca2+]i)的變化帶來(lái)的影響，以探討caveolin-1/caveolae在腦血管痙攣中的可能作用。 方法： 1、分離SD大鼠頸總動(dòng)脈，利用組織貼塊法體外培養(yǎng)血管平滑肌細(xì)胞，通過(guò)對(duì)細(xì)胞形態(tài)學(xué)觀察和平滑肌細(xì)胞特異性α-肌動(dòng)蛋白(α-actin)免疫熒光染色進(jìn)行鑒定。 2、蛋白印跡Western blot法檢測(cè)1mmol/L、2.5mmol/L和5mmol/L濃度MβCD溶液處理后VSMCs caveolin-1表達(dá)水平。 3、蛋白印跡Western blot法檢測(cè)20ug/L、50ug/L、100ug/L濃度TNF-α溶液孵育后VSMCs caveolin-1表達(dá)水平。 4、通過(guò)MβCD(5mmol/L)和炎癥因子TNF-α(100ug/L)的不同處理將細(xì)胞分為四組：A.對(duì)照組；B. MβCD組；C. TNF-α組；D. TNF-α+MβCD組。采用TILL熒光鈣成像系統(tǒng)檢測(cè)活細(xì)胞在ET-1(100nmol/L)刺激下胞內(nèi)鈣熒光強(qiáng)度(FI)變化情況。 結(jié)果： 1、經(jīng)過(guò)對(duì)細(xì)胞形態(tài)學(xué)觀察和α-actin免疫熒光染色鑒定，原代成功分離和培養(yǎng)大鼠頸總動(dòng)脈VSMCs。 2、在不同濃度MβCD作用下使細(xì)胞caveolin-1蛋白含量隨MβCD濃度的升高而降低。caveolin-1蛋白含量5mmol/L MβCD處理組較對(duì)照組下降明顯(P0.01)。 3、在不同濃度TNF-α作用下使細(xì)胞caveolin-1蛋白含量隨TNF-α濃度的升高而升高。caveolin-1蛋白含量100ug/L濃度TNF-α處理組較對(duì)照組上升明顯(P0.01)。 4、TILL熒光鈣成像系統(tǒng)檢測(cè)四組細(xì)胞結(jié)果顯示1)、各組細(xì)胞靜息狀態(tài)R0分別為A:0.5916±0.0820,B:0.5753±0.0790, C:0.6551±0.0837,D:0.6105±0.0804,四間細(xì)胞組間無(wú)顯著差異(P0.05)。2)、四組細(xì)胞經(jīng)相同刺激后胞內(nèi)鈣熒光強(qiáng)度(FI)分別較靜息狀態(tài)上升,A組上升(115±17)％；B組上升(67±15)％，，較A組差異顯著(P0.05)；C組上升(279±21)％,較A組差異顯著(P0.01)；D組上升(88±16)％,較C組差異顯著(P0.01)。 結(jié)論： 1、5mmol/L MβCD能有效干擾破壞細(xì)胞膜caveolae結(jié)構(gòu)，可顯著下調(diào)VSMCscaveolin-1蛋白表達(dá)。 2、100ug/L TNF-α孵育可顯著上調(diào)VSMCs caveolin-1蛋白表達(dá)。 3、caveolin-1蛋白表達(dá)上調(diào)后可明顯增加ET-1誘導(dǎo)的VSMCs胞內(nèi)鈣濃度升高程度，而MβCD有效干擾破壞caveolae結(jié)構(gòu)后導(dǎo)致caveolin-1蛋白表達(dá)下調(diào)后，可明顯降低ET-1誘導(dǎo)的VSMCs胞內(nèi)鈣濃度升高程度，caveolin-1/caveolae可能是抑制腦血管痙攣發(fā)生的重要靶點(diǎn)。
[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.
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本文編號(hào)：1844288