ANXA2在视网膜新生血管形成过程中的表达及作用机制

发布时间：2018-04-03 19:24

本文选题：膜联蛋白A2　切入点：氧诱导　出处：《青岛大学》2012年博士论文

【摘要】：目的通过观察ANXA2在体外高糖培养的人脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVEC)及氧诱导C57BL/6J小鼠视网膜新生血管中的表达,研究ANXA2对HUVEC增殖,迁移,成管能力的影响,对小鼠视网膜新生血管发育的影响,以及对新生血管相关因子表达的调控,探讨ANXA2在视网膜新生血管形成过程中的作用和机制。方法 (1)体外实验应用HUVEC细胞,实验分四组：正常组(Normal,常规培养),高糖培养下的高糖对照组(Mock,无处理)、阴性干扰组(SiControl)、阳性干扰组(SiANXA2)。应用Real-time PCR和Western blot方法检测各实验组HUVEC中膜联蛋白A2 (ANXA2)基因及蛋白的表达情况,MTT方法检测各组0h,24h,48h和72h细胞增殖能力,划痕实验观察各实验组细胞迁移能力,成管实验评估细胞的成管能力。并通过Real-time PCR检测各实验组中新生血管相关因子VEGFA,MMP2,MMP9,TIMP2的mRNA表达情况。 (2)体内实验应用C57BL/6J小鼠。小鼠出生后7天,放入氧浓度75-80%氧箱内5天后取出正常环境下饲养,构建氧诱导(OIR)小鼠模型。实验分四组：正常组(Normal,正常饲养),OIR空白对照组(Mock,无处理),OIR阴性干扰组(SiANXA2_M)、和OIR阳性干扰组(SiANXA2)。分别取正常组和OIR空白对照组出生后12,13,14,15,16,17,21,30天小鼠,异硫氰酸标记的右旋糖酐(FD-2000S)行上腔静脉灌注,视网膜铺片,荧光显微镜对比观察氧诱导视网膜新生血管形态学变化,应用Real-time PCR检测对比两组间相应日龄小鼠视网膜ANXA2 mRNA的表达变化。选择小鼠出生后17天(P17)时间点,分别提取4组中小鼠视网膜标本,通过视网膜铺片方法对比观察视网膜新生血管形态学变化,Real-time PCR及Western blot检测各组中ANXA2的表达及ANXA2基因干扰后新生血管相关因子Vegfa,Mmp2,Mmp9, Timp2的表达变化。应用SPSS软件对数据行统计学处理,应用单因素方差分析进行组间比较(P0.05)。结果 (1)体外实验：高糖对照组HUVEC中ANXA2表达显著高于正常组(P0.05)。高糖对照组HUVEC的增殖,细胞迁移数量(102.3±4.9 VS 50.3±2.5)和成管数量(17.3±1.5 VS 10±1.4)以及VEGFA, MMP2,MMP9和TIMP2的mRNA表达均显著高于正常组(P0.05)。基因干扰后,在基因和蛋白水平均表明ANXA2在阴性干扰组表达无明显改变,在阳性干扰组表达有明显下降,SiANXA2可以成功干扰ANXA2基因的表达。ANXA2基因表达下降后,HUVEC的增殖、细胞迁移数量(41.1±1.5 VS 102.3±4.9)和成管数量(7.6±1.2 VS 17.3±1.5)显著下降,VEGFA,MMP2,MMP9,TIMP2表达明显下调(P0.05)。 (2)体内实验：ANXA2在视网膜血管发育旺盛期高表达,在平缓期低表达。P17小鼠视网膜铺片发现,与OIR空白对照组和OIR阴性干扰组相比,OIR阳性干扰组视网膜新生血管迂曲现象减轻,血管渗漏较少,微血管瘤及新生血管芽减少。视网膜层次较明显。OIR空白对照组ANXA2表达显著高于正常组(P0.05)。ANXA2基因沉默后,ANXA2在OIR阳性干扰组表达有明显下降(P0.05)。Vegfa,Mmp2,Mmp9在OIR空白对照组较正常组显著上调,在OIR阳性干扰组较OIR空白对照组显著下调(P0.05)。结论ANXA2在高糖培养的HUVEC和氧诱导小鼠视网膜新生血管中均高表达,与血管内皮细胞内皮细胞增殖,迁移和成管能力相关,可调节新生血管相关因子的表达,参与视网膜新生血管的形成。RNA干扰技术能成功抑制ANXA2基因的表达,ANXA2有望成为防治视网膜新生血管新的靶点。
[Abstract]:Objective To observe the effect of ANXA2 on high glucose in vitro cultured human umbilical vein endothelial cells (Human umbilical vein endothelial cells, HUVEC) C57BL/6J expression of retinal neovascularization in mice induced by ANXA2 and oxygen, on the migration of HUVEC proliferation, tube formation effect ability, influence on the development of retinal neovascularization in mice, and the regulation of the expression of related factors on the neovascularization, formation of ANXA2 effect and mechanism in the process of retinal neovascularization.
Method
(1) the application of HUVEC cells in vitro were divided into four groups: normal group (Normal, cultured under high glucose), high glucose group (Mock treatment), the negative interference group (SiControl), the positive interference group (SiANXA2). Annexin A2 detected by Real-time PCR and Western blot method the experimental group HUVEC (ANXA2) gene and protein expression of MTT was detected in 0h, 24h, 48h and 72h cell proliferation, observed in each experimental group cell migration scratch test, experimental evaluation of cell into a tube into the tube. And through the Real-time PCR detection of neovascularization in each experimental group related factor VEGFA MMP2, MMP9, mRNA, TIMP2 expression.
(2) in vivo experiments using C57BL/6J mice. Mice 7 days after birth, 75-80% into the oxygen concentration of oxygen inside take out after 5 days under normal circumstances were constructed with oxygen induced (OIR) mouse model. The experiment was divided into four groups: normal group (Normal, normal feeding), OIR control group (Mock, no treatment), OIR the negative interference group (SiANXA2_M), and OIR positive interference group (SiANXA2) respectively. Normal group and OIR control group 12,13,14,15,16,17,21,30 days after birth in mice, fluorescein isothiocyanate dextran (FD-2000S) for superior vena cava perfusion, retinal flatmount, fluorescence microscopy observation changes of retinal neovascularization in oxygen induced morphological changes in the expression of. Comparison and application of Real-time PCR in two groups were detected between the corresponding day old mouse retinal ANXA2 mRNA. Mice 17 days after birth (P17) time points were extracted from the 4 groups in the mouse retina retinal flatmount specimens, through comparative observation method Changes of retinal neovascularization morphology, ANXA2 gene and expression of ANXA2 Real-time PCR and Western blot interference were detected in the angiogenesis related factors Vegfa, Mmp2, Mmp9, Timp2 expression changes. Application of SPSS software for data processing, using single factor analysis of variance were compared between the groups (P0.05).
Result
(1) in vitro: high glucose control group HUVEC ANXA2 expression was significantly higher than the normal group (P0.05). The proliferation of high glucose control group HUVEC, the number of cell migration (102.3 + 4.9 VS 50.3 + 2.5) and the number of tubes (17.3 + 1.5 VS 10 + 1.4), VEGFA, MMP2, MMP9 and TIMP2 mRNA DA were significantly higher than the normal group (P0.05). After gene interference, in gene and protein levels showed that ANXA2 in the negative interference group showed no significant change in the positive interference group was significantly decreased, SiANXA2 can be successfully interfered with the expression of.ANXA2 gene ANXA2 gene expression decreased after HUVEC cells proliferation, migration (41.1. 1.5 VS 102.3 + 4.9) and the number of tubes (7.6 + 1.2 VS 17.3 + 1.5) decreased significantly, VEGFA, MMP2, MMP9, TIMP2 gene expression was significantly decreased (P0.05).
(2) in vivo: high expression of ANXA2 in retinal vascular development in the period of vigorous period of low expression of.P17 mice retinas, OIR and blank control group and OIR group compared with the negative interference, reduce OIR positive interference group, retinal neovascularization is tortuous vascular leakage phenomenon, fewer microaneurysms and new vascular buds decreased the retina is obvious..OIR control group the expression of ANXA2 was significantly higher than the normal group (P0.05) after.ANXA2 gene silencing, ANXA2 expression was significantly decreased in OIR positive interference group (P0.05).Vegfa, Mmp2, Mmp9 group compared with normal group were significantly up-regulated in OIR gap in OIR positive interference group compared with the OIR control group was significantly reduced (P0.05).
Conclusion ANXA2 induced retinal neovascularization in mice were highly expressed in HUVEC cultured with high glucose and oxygen, and endothelial cell proliferation, migration and tube formation ability, can adjust the expression of angiogenesis related factors, the formation of.RNA interference can successfully inhibit the expression of ANXA2 gene in retinal neovascularization, ANXA2 is expected to become the target the prevention and treatment of retinal neovascularization.

【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R774.1

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