CAV-1对人脐静脉内皮细胞中MCP-1表达的影响

发布时间：2018-03-15 02:13

本文选题：动脉粥样硬化　切入点：氧化低密度脂蛋白　出处：《华中科技大学》2008年硕士论文　论文类型：学位论文

【摘要】： 单核细胞趋化蛋白-1(MCP-1)的表达增加是内皮功能紊乱的重要表现之一,氧化低密度脂蛋白(ox-LDL)可以通过血凝素样氧化低密度脂蛋白受体(LOX-1)介导内皮细胞MCP-1的表达增加,若预先加入反义LOX-1 mRNA则可抑制上述过程,表明LOX-1在介导ox-LDL诱导的内皮细胞中MCP-1表达的信号转导途径中发挥重要作用。然而LOX-1的结构分析显示LOX-1胞内段无SH2域,且不与G蛋白偶联,却可激活酪氨酸蛋白激酶途径及G蛋白介导的PKC信号途径。因此,LOX-1如何介导信号跨膜转导的机制尚不清楚。由于多种信号分子聚集在Caveolae区,CAV-1是其信号转导中心,可介导多种受体的信号转导,且本课题组通过激光共聚焦检测到LOX-1与CAV-1在中国仓鼠卵巢细胞(CHO)上共定位,由此我们推测,CAV-1可能协助LOX-1介导ox-LDL诱导内皮细胞MCP-1的表达增加。本实验采用激光共聚焦技术,检测到培养的人脐静脉内皮细胞中LOX-1与CAV-1共定位。采用RT-PCR和Western blot检测cav-1 mRNA及蛋白水平,以此筛选出一对有效的特异性cav-1 siRNA。在上述基础上,将培养的人脐静脉内皮细胞分为以下三组:空白对照组(正常培养基),ox-LDL刺激组(未转染特异性cav-1 siRNA)及cav-1 siRNA组(转染体外化学合成的特异性cav-1 siRNA24 h后ox-LDL刺激24 h),采用RT-PCR和ELISA方法检测MCP-1 mRNA及蛋白水平的变化。结果发现与空白对照组相比,ox-LDL刺激组MCP-1的mRNA和蛋白表达量显著增加(P0.05);而cav-1 siRNA组可显著降低ox-LDL诱导的MCP-1的表达量(P0.05)。我们的结果表明有效抑制CAV-1的表达可以下调ox-LDL诱导的内皮细胞MCP-1的表达,提示CAV-1在ox-LDL诱导内皮功能紊乱的信号传递途径中可能发挥重要作用。我们将进一步采用突变体构建、FRET等技术证实CAV-1在LOX-1信号传递中的介导作用。
[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.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363

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