窖蛋白-1对大鼠星形胶质细胞糖氧剥夺损伤的保护作用

发布时间：2018-06-08 05:00

本文选题：窖蛋白-1 + 星形胶质细胞　；参考：《南京大学》2014年硕士论文

【摘要】：目的：胞膜窖(caveolae)是细胞膜表面的微小凹陷区域,窖蛋白-1(caveolin-1,Cav-1)是胞膜窖的主要结构蛋白。既往研究报道指出Cav-1参与了缺血性脑卒中后细胞信号传导的调节,对中枢神经系统具有保护作用,但是这一保护机制目前仍不清楚。本研究旨在探讨Cav-1对体外培养的大鼠星形胶质细胞糖氧剥夺(oxygen-glucose deprivation, OGD)损伤的影响极其作用机制。方法：原代培养出生24 h内的SD大鼠大脑皮质星形胶质细胞,建立OGD损伤模型,将细胞分为对照组和OGD组,OGD组给予星形胶质细胞6h的OGD和24 h的再灌注损伤。用免疫荧光的方法检测星形胶质细胞Cav-1表达,实时荧光定量PCR和Western blot的方法检测星形胶质细胞OGD后24 h Cav-1表达的变化。根据小干扰RNA (small interference RNA, siRNA)的原理,将Cav-1 siRNA转染入星形胶质细胞,抑制Cav-1在星形胶质细胞中的表达,或给予外源性窖蛋白-1脚手架区肽段(caveolin-1 scaffolding domain peptide, C1-SD82-101)增强细胞中Cav-1的表达后,采用细胞计数试剂盒-8 (cell counting kit-8, CCK-8)的方法检测OGD损伤前后星形胶质细胞的存活率,采用Western blot的方法检测细胞中细胞外信号调节激酶1/2 (extracellular signal-regulated kinase 1/2, ERK1/2)及磷酸化细胞外信号调节激酶1/2 (phosphorylated extracellular signal-regulated kinase 1/2, pERK1/2)的水平。使用MEK抑制剂(U0126)抑制细胞中ERK1/2信号通路后采用CCK-8的方法检测星形胶质细胞存活率。结果：Cav-1在大鼠大脑皮质星形胶质细胞普遍表达。与对照组相比,OGD组星形胶质细胞Cav-1表达量下降(P0.05)。抑制星形胶质细胞中Cav-1的表达后,与非OGD组相比,OGD组星形胶质细胞存活率下降(P0.05)。给予外源性C1-SD82-101(10uM)增强星形胶质细胞内Cav-1的表达后,与对照组相比,OGD组星形胶质细胞存活率升高(P0.05)。同时我们发现,给予外源性C1-SD82-101组与未给予C1-SD82-101组相比,OGD后细胞存活率升高,并且细胞内pERK1/2表达明显升高,然而给予MEK抑制剂(U0126)后,Cav-1对星形胶质细胞的保护作用不再存在。结论：Cav-1对SD大鼠大脑皮质星形胶质细胞存在保护作用,这一保护作用机制可能与Cav-1参与了OGD后星形胶质细胞ERK1/2信号通路的调节有关。
[Abstract]:Objective: caveolae1 is a small hollow area of cell membrane surface and Cav-1 is the main structural protein of cellar. Previous studies have shown that Cav-1 is involved in the regulation of cellular signal transduction after ischemic stroke and has a protective effect on the central nervous system, but this protective mechanism is still unclear. The aim of this study was to investigate the effects of Cav-1 on oxygen-glucose deprivation (OGDN) injury of cultured rat astrocytes in vitro. Methods: primary cultured cerebral cortex astrocytes of SD rats within 24 hours were cultured and OGD injury models were established. The cells were divided into two groups: control group and OGD group. Astrocytes were given OGD for 6 h and reperfusion injury for 24 h in OGD group. The expression of Cav-1 in astrocytes was detected by immunofluorescence, and the changes of Cav-1 expression in astrocytes at 24 h after OGD were detected by real-time fluorescence quantitative PCR and Western blot. According to the principle of small interfering RNAs, Cav-1 siRNA was transfected into astrocytes to inhibit the expression of Cav-1 in astrocytes, or exogenous cellar protein 1 scaffold region peptide fragment caveolin-1 scaffolding domain peptide, C1-SD82-101) enhanced the expression of Cav-1 in astrocytes. The survival rate of astrocytes before and after OGD-induced injury was detected by cell count kit -8 cell counting kit-8 (CCK-8). The levels of extracellular signal-regulated kinase 1 / 2 (ERK1 / 2) and phosphorylated extracellular signal-regulated kinase 1 / 2 (pERK1 / 2) were detected by Western blot. MEK inhibitor U0126) was used to inhibit ERK1 / 2 signaling pathway and then CCK-8 was used to detect the survival rate of astrocytes. Results: Cav-1 was widely expressed in astrocytes of cerebral cortex of rats. Compared with the control group, the expression of Cav-1 in astrocytes decreased in OGD group. After inhibiting the expression of Cav-1 in astrocytes, the survival rate of astrocytes in OGD group was lower than that in non-OGD group. Exogenous C1-SD82-101UM) enhanced the expression of Cav-1 in astrocytes, and the survival rate of astrocytes in OGD group was higher than that in control group (P 0.05). At the same time, we found that the survival rate and the expression of pERK1 / 2 in exogenous C1-SD82-101 group were higher than those in non-C1-SD82-101 group, but the protective effect of Cav-1 on astrocytes was no longer present after MEK inhibitor (U0126). Conclusion: Cav-1 has protective effect on astrocytes in cerebral cortex of SD rats. This protective mechanism may be related to the regulation of ERK1 / 2 signaling pathway in astrocytes after OGD.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3

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