發(fā)布時(shí)間：2018-03-15 02:13

  本文選題：動脈粥樣硬化　切入點(diǎn)：氧化低密度脂蛋白　出處：《華中科技大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 單核細(xì)胞趨化蛋白-1(MCP-1)的表達(dá)增加是內(nèi)皮功能紊亂的重要表現(xiàn)之一,氧化低密度脂蛋白(ox-LDL)可以通過血凝素樣氧化低密度脂蛋白受體(LOX-1)介導(dǎo)內(nèi)皮細(xì)胞MCP-1的表達(dá)增加,若預(yù)先加入反義LOX-1 mRNA則可抑制上述過程,表明LOX-1在介導(dǎo)ox-LDL誘導(dǎo)的內(nèi)皮細(xì)胞中MCP-1表達(dá)的信號轉(zhuǎn)導(dǎo)途徑中發(fā)揮重要作用。然而LOX-1的結(jié)構(gòu)分析顯示LOX-1胞內(nèi)段無SH2域,且不與G蛋白偶聯(lián),卻可激活酪氨酸蛋白激酶途徑及G蛋白介導(dǎo)的PKC信號途徑。因此,LOX-1如何介導(dǎo)信號跨膜轉(zhuǎn)導(dǎo)的機(jī)制尚不清楚。由于多種信號分子聚集在Caveolae區(qū),CAV-1是其信號轉(zhuǎn)導(dǎo)中心,可介導(dǎo)多種受體的信號轉(zhuǎn)導(dǎo),且本課題組通過激光共聚焦檢測到LOX-1與CAV-1在中國倉鼠卵巢細(xì)胞(CHO)上共定位,由此我們推測,CAV-1可能協(xié)助LOX-1介導(dǎo)ox-LDL誘導(dǎo)內(nèi)皮細(xì)胞MCP-1的表達(dá)增加。 本實(shí)驗(yàn)采用激光共聚焦技術(shù),檢測到培養(yǎng)的人臍靜脈內(nèi)皮細(xì)胞中LOX-1與CAV-1共定位。采用RT-PCR和Western blot檢測cav-1 mRNA及蛋白水平,以此篩選出一對有效的特異性cav-1 siRNA。在上述基礎(chǔ)上,將培養(yǎng)的人臍靜脈內(nèi)皮細(xì)胞分為以下三組:空白對照組(正常培養(yǎng)基),ox-LDL刺激組(未轉(zhuǎn)染特異性cav-1 siRNA)及cav-1 siRNA組(轉(zhuǎn)染體外化學(xué)合成的特異性cav-1 siRNA24 h后ox-LDL刺激24 h),采用RT-PCR和ELISA方法檢測MCP-1 mRNA及蛋白水平的變化。結(jié)果發(fā)現(xiàn)與空白對照組相比,ox-LDL刺激組MCP-1的mRNA和蛋白表達(dá)量顯著增加(P0.05);而cav-1 siRNA組可顯著降低ox-LDL誘導(dǎo)的MCP-1的表達(dá)量(P0.05)。 我們的結(jié)果表明有效抑制CAV-1的表達(dá)可以下調(diào)ox-LDL誘導(dǎo)的內(nèi)皮細(xì)胞MCP-1的表達(dá),提示CAV-1在ox-LDL誘導(dǎo)內(nèi)皮功能紊亂的信號傳遞途徑中可能發(fā)揮重要作用。我們將進(jìn)一步采用突變體構(gòu)建、FRET等技術(shù)證實(shí)CAV-1在LOX-1信號傳遞中的介導(dǎo)作用。
[Abstract]:The increased expression of monocyte chemoattractant protein (MCP-1) is one of the important manifestations of endothelial dysfunction. Oxidized low density lipoprotein (ox-LDL) can induce the increase of MCP-1 expression in endothelial cells through hemagglutinin oxidized low density lipoprotein receptor (LOX-1). Adding antisense LOX-1 mRNA in advance can inhibit the above process, suggesting that LOX-1 plays an important role in the signal transduction pathway of MCP-1 expression in endothelial cells induced by ox-LDL. However, the structural analysis of LOX-1 shows that there is no SH2 domain in the intracellular region of LOX-1. And not coupled to G protein, However, tyrosine protein kinase pathway and G protein-mediated PKC signaling pathway can be activated. It is unclear how LOX-1 mediates signal transmembrane transduction. Because many signaling molecules gather in the Caveolae region, CAV-1 is the signal transduction center. The co-localization of LOX-1 and CAV-1 in Chinese hamster ovary cells was detected by confocal laser. We speculate that CAV-1 may assist LOX-1 to induce the increase of MCP-1 expression in endothelial cells induced by ox-LDL. The co-localization of LOX-1 and CAV-1 in cultured human umbilical vein endothelial cells was detected by laser confocal technique. The levels of cav-1 mRNA and protein in cultured human umbilical vein endothelial cells were detected by RT-PCR and Western blot. The cultured human umbilical vein endothelial cells were divided into the following three groups: blank control group (normal medium) (non-transfection specific cav-1 siRNAs) and cav-1 siRNA group (ox-LDL stimulated 24 h after transfection of chemically synthesized specific cav-1 siRNA24 h). The changes of MCP-1 mRNA and protein levels were detected by RT-PCR and ELISA. The results showed that the expression of mRNA and protein in MCP-1 stimulated by ox-LDL was significantly higher than that in control group, while that in cav-1 siRNA group was significantly lower than that in control group. Our results suggest that inhibiting the expression of CAV-1 can down-regulate the expression of MCP-1 in endothelial cells induced by ox-LDL. It is suggested that CAV-1 may play an important role in the signal transduction pathway of endothelial dysfunction induced by ox-LDL. We will further confirm the role of CAV-1 in LOX-1 signal transduction by using mutants to construct fret and other techniques.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R363

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本文編號：1613967