G蛋白信号转导调节子5参与实验性脉络膜新生血管及其可能的机制

发布时间：2018-04-27 12:46

本文选题：脉络膜新生血管 + G蛋白信号转导调节子5　；参考：《第四军医大学》2010年硕士论文

【摘要】：研究背景脉络膜新生血管(CNV)是眼内新生血管的重要表现形式之一,常导致严重的视力丧失,其生成是一个十分复杂的过程,受多种因素或因子精细而敏感的调控。Bruch膜损伤和缺氧是CNV形成的重要机制,前者可降低营养物质运输能力和增加脂质沉积,导致Bruch膜发生断裂,新生血管通过断裂的Bruch膜长入视网膜下间隙;后者可诱导视网膜色素上皮(RPE)细胞数量和功能改变,释放多种细胞因子,导致RPE-Bruch膜-脉络膜毛细血管复合体完整性破坏,促进内皮细胞增生和移行,最终形成新生血管。 G蛋白信号转导调节子5(RGS5)属于RGS蛋白超家族中的R4/B亚科,不仅负向调控G蛋白偶联受体相结合的信号转导,还能抑制该通路下游的酪氨酸激酶激活小G蛋白/丝裂原激活蛋白激酶(MAPK)信号通路的激活,与缺氧及病理性新生血管形成密切相关,且这一作用可能与血管内皮生长因子(VEGF)有关。VEGF是RPE细胞分泌的因子之一,在CNV的发生中起着重要的作用。贝伐单抗(Avastin)作为抗VEGF重组单克隆抗体,能阻断VEGF-A所有亚型与内皮细胞表面受体的结合,通过抑制VEGF活性,达到有效阻碍CNV形成的目的。目的探讨RGS5参与体内实验性CNV生成及在体外缺氧环境下RPE细胞中表达的可能机制。方法(1)532nm激光诱导88只(176眼)雄性棕色挪威(BN)大鼠实验性CNV。首先按时间分为光凝后1d(n=6)、3d(n=6)、7d(n=7)和14d(n=21)四组,观察RGS5及VEGF的表达;选取光凝7d(n=12)和14d(n=36)作观察点,按处理因素分为生理盐水组及Avastin注射组,观察通过特异性抑制VEGF表达对RGS5表达产生的影响;(2)在正常培养的人RPE细胞培养基中加入终浓度为200μM的CoCl2建立细胞化学缺氧模型。按缺氧时间分为0、1、3、6、12和24h六组,观察RGS5和VEGF表达的变化;另选取12h和24h作观察点,按处理因素分为缺氧对照组和Avastin处理组,Avastin包括25μg/ml、100μg/ml和250μg/ml三种浓度,观察抑制VEGF表达后RGS5表达的变化。采用组织病理学切片和脉络膜铺片分别观察CNV厚度和面积;免疫荧光染色法、Western blot和RT-PCR分别检测RGS5和VEGF蛋白与mRNA的表达。采用Student’s t-检验进行统计学分析。结果(1)光凝后1d、3d、7d和14d,RGS5蛋白及mRNA相对表达量均于1d(t_(1d)=3.451,P 0.05;t_(1d)=3.646,P 0.05)和3d(t_(3d)=3.085,P 0.05;t_(3d)=3.888,P 0.05)下降,7d开始上升,14d(t_(14d)=2.079,P 0.05;t_(14d)=2.194,P 0.05)达到高峰;VEGF蛋白及mRNA相对表达量均于7d(t_(7d)=7.959,P 0.01;t_(7d)=7.959,P 0.01)达到高峰,14d有所下降,但仍高于正常水平。RGS5表达高峰晚于VEGF出现;玻璃体腔注射Avastin后,CNV生成的厚度和面积均减小(t=2.616,P 0.05;t=15.179,P = 0.000),光凝后7d VEGF蛋白和mRNA的表达及光凝后14d RGS5蛋白和mRNA的表达均下调;(2)在上述结果基础上,选择RPE细胞进行体外实验得到,正常条件下RGS5主要表达于人RPE细胞胞浆中,与VEGF存在共表达区域。随着缺氧时间的延长,RPE细胞中RGS5蛋白及mRNA表达逐渐上调,24h时表达达高峰(0.932±0.104,t_(24)=3.106,P=0.011;0.742±0.083,t_(24)=2.852,P=0.017);VEGF蛋白及mRNA表达12h达到高峰(1.022±0.141,t_(12)=4.144,P=0.002;0.491±0.063,t_(12)=5.707,P=0.000),24h有所下降,但仍高于未缺氧的RPE细胞(0.942±0.125,t_(24)=3.306,P=0.008;0.425±0.080,t_(24)=3.239,P=0.011)。浓度为100μg/ml和250μg/ml的Avastin有效抑制VEGF蛋白(t_(100)=2.794,P=0.019;t_(250)=3.396,P=0.007)和mRNA(t_(100)=2.823,P=0.018;t_(250)=9.584,P=0.000)表达后,RGS5蛋白(t_(100)=2.953, P=0.014;t_(250)=2.913, P=0.015)和mRNA(t_(100)=3.009, P=0.013;t_(250)=4.243, P=0.002)表达受到抑制。结论RGS5与VEGF表达和CNV生成相关,且两者表达有时相关系;通过抑制VEGF表达有效抑制CNV过程中,RGS5表达下调。在此基础上体外实验结果提示,正常条件下RGS5表达于RPE细胞胞浆中,与VEGF存在共表达区域;RGS5和VEGF的表达具有时相关系,通过抑制VEGF,RGS5表达随之降低。综上提示RGS5可能位于VEGF信号通路下游,参与了VEGF相关的CNV生成机制,且与CNV发生相关的RGS5表达可能部分是RPE细胞源性的。
[Abstract]:Background Choroidal neovascularization ( CNV ) is one of the important manifestations of neovascularization in the eye , which often leads to severe loss of vision . It is a very complex process which is sensitive to various factors or factors . Bruch membrane damage and hypoxia are important mechanisms for CNV formation .

The G protein signal transduction regulator 5 ( RGS5 ) belongs to the R4 / B subfamily in the RGS protein superfamily , which not only negatively regulates the activation of the G protein coupled receptor , but also inhibits the activation of the tyrosine kinase activated small G protein / mitogen - activated protein kinase ( MAPK ) signal pathway downstream of the pathway , and plays an important role in the pathogenesis of CNV .

Objective To investigate the possible mechanism of RGS5 in the generation of experimental CNV and the expression of RGS5 in RPE cells in vitro .

Methods ( 1 ) The expression of RGS5 and VEGF was observed in 88 ( 176 eyes ) of male brown norway ( BN ) rats induced by 532 nm laser . The expression of RGS5 and VEGF was observed .

Results ( 1 ) The relative expression levels of RGS5 protein and mRNA in RPE cells decreased after 7 days ( t _ ( 1d ) = 3.451 , P 0.05 ; t _ ( d ) = 3.646 , P 0.05 ; t _ ( d ) = 3.085 , P 0.05 ; t _ ( 3d ) = 3.888 , P = 0.000 ) . t _ ( 24 ) = 3.106 , P = 0 . 011 ; 0 . 742 卤 0 . 083 , t _ ( 24 ) = 2.852 , P = 0 . 017 ) ; VEGF protein and mRNA expression peaked at 12 h ( 1.022 卤 0 . 141 , t _ ( 12 ) = 4.144 , P = 0.002 ; 0 . 491 卤 0.063 , t _ ( 12 ) = 5.707 , P = 0.000 ) , but still higher than that of uninjured RPE cells ( 0.942 卤 0.125 , t _ ( 24 ) = 3.306 , P = 0.008 ; 0.425 卤 0.080 , t _ ( 24 ) = 3.239 , P = 0 . 011 ) . The expression of VEGF protein ( t _ ( 100 ) = 2.794 , P = 0.019 ; t _ ( 250 ) = 3.396 , P = 0.007 ) and mRNA ( t _ ( 100 ) = 2.823 , P = 0 . 018 ; t _ ( 250 )) = 2.823 , P = 0 . 014 ; t _ ( 250 ) = 2.913 , P = 0 . 015 ) and mRNA ( t _ ( 100 ) = 3.009 , P = 0 . 013 ; t _ ( 250 ) = 4.243 , P = 0.002 ) were inhibited .

Conclusion RGS5 is related to the expression of VEGF and CNV , and the expression of RGS5 is downregulated by inhibiting the expression of VEGF . In vitro , RGS5 is expressed in the cytoplasm of RPE cells , and the expression of RGS5 and VEGF is decreased .

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R773.4

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