Robo4在调节血管内皮细胞通透性中的作用及机制研究

发布时间：2018-06-23 08:39

本文选题：Roundabout4(Robo4) + 小干扰RNA　；参考：《吉林大学》2013年博士论文

【摘要】：血-视网膜屏障是存在于血液和视网膜之间具有限制性通透性的结构，对维护视网膜组织微环境的稳定具有重要意义。血-视网膜屏障包括内屏障和外屏障。血-视网膜内屏障主要由视网膜毛细血管内皮细胞及细胞间的紧密连接构成，血管内皮细胞是内屏障的结构基础。血管内皮屏障功能障碍导致血管渗漏是糖尿病视网膜病变、早产儿视网膜病变、Coats病等疾病发生的主要病理改变之一，也是许多视网膜血管性疾病视力损害的重要原因。因此，探索内皮屏障功能损害发生机理具有十分重要的临床意义。血管内皮通透性增高可能有两种机制，一种是细胞内路径（transcellular pathway），另一种是细胞旁路径（paracellular pathway）。细胞旁路径是内皮细胞渗透性增高的主要途径，内皮细胞间广泛的紧密连接（tight junction, TJ）是细胞旁路径的结构基础。多项研究表明，occludin和ZO-1是两个关键的紧密连接蛋白。TJ蛋白表达量的变化、在细胞内的位置分布变化和／或结构功能的异常均可能破坏TJ的完整性，引起细胞间连接的开放，导致血管内皮通透性增高。研究发现，内皮细胞骨架，特别是肌动蛋白（actin）的重组和再分布也是导致内皮细胞收缩、血管通透性增高的主要病理基础。 Robo家族(Roundabout family)是一类与发育有关的保守跨膜蛋白家族，目前已知其对神经轴突延伸和神经细胞迁移有重要的调控作用。新近发现的Robo4，是Robo家族的第四个成员，又称为MRB (magic roundabout)，是一种保守跨膜受体蛋白。Robo4与其他Robo蛋白质不同，只特异性表达于血管新生活跃的内皮细胞表面，并参与调控内皮细胞的迁移。最近有文献报道Robo4可以阻断血管内皮生长因子（vascularendothelial growth factor, VEGF）下游的信号途径而抑制病理性新生血管的发生，同时在缺氧视网膜病变动物模型中发现，Robo4可以明显降低视网膜血管的渗漏性。多数学者认为Robo4可以降低内皮细胞的通透性，维持成熟血管系统的稳定，但关于Robo4在视网膜内皮细胞通透性的确切作用机制尚未见相关报道。通过观察Robo4对紧密连接蛋白及细胞骨架肌动蛋白actin的影响，探讨Robo4对视网膜内皮细胞通透性的调控作用，并对可能的机制进行深入探讨。研究分为三部分。首先，在应用糖基化终末产物（advanced glycation end product, AGE）诱导内皮细胞通透性增加的过程中检测Robo4蛋白的表达变化，结果显示：随着AGE浓度的增加，Robo4蛋白的表达呈下降趋势，呈剂量依赖性；随着AGE作用时间的延长，Robo4蛋白的表达呈下降趋势，呈时间依赖性，表明内皮细胞通透性增加的过程中伴随着Robo4蛋白表达的减少，提示Robo4蛋白可能在视网膜内皮细胞通透性的调控过程中发挥作用；为进一步证实Robo4对视网膜通透性的调控作用，应用Robo4小干扰RNA特定沉默Robo4的表达，通过测定葡聚糖的荧光发射强度评价视网膜血管内皮细胞单层通透性的变化，结果表明：干扰抑制视网膜血管内皮细胞中Robo4的表达水平，会使内皮细胞的通透性增加，破坏内皮细胞的屏障功能。由于紧密连接相关蛋白与细胞骨架肌动蛋白之间的相互作用在维持紧密连接的完整性方面具有重要作用，推测在抑制Robo4表达增加内皮细胞通透性的过程中，可能伴随着细胞间紧密连接的开放，Robo4可能通过改变紧密连接从而发挥对内皮细胞通透性的调节作用。在第二部分研究中，应用western blot检测紧密连接蛋白ZO-1、occludin的表达变化，结果显示：干扰抑制视网膜血管内皮细胞中Robo4蛋白的表达，可相应降低细胞中occludin和ZO-1蛋白的表达水平；同时应用罗丹明-鬼笔环肽对细胞内的细胞骨架肌动蛋白F-actin进行染色发现，胞浆内出现大量弥散分布的应力纤维，而分布在细胞周边的外周致密束则明显减少，表明靶向敲除Robo4基因的表达可以促使紧密连接开放，从而导致体外培养的内皮细胞单层通透性增加，其紧密连接的开放可能与紧密连接相关蛋白occludin、ZO-1的表达减少及细胞骨架肌动蛋白的重组和再分布密切相关，初步阐明Robo4调控内皮细胞通透性的机制。 F-actin的组装和去组装受到细胞质内多种蛋白的调节，称为肌动蛋白结合蛋白。目前已发现了100多种肌动蛋白结合蛋白，其中肌动蛋白解聚因子/丝切蛋白（actindepolymerizing factor/cofilin, ADF/cofilin）被认为有着关键作用，是近年来研究的热点。在第三部分研究中，应用western blot和siRNA共转染技术，阐明LIM激酶（LIM kinase,LIMK）-cofilin信号途径参与了Robo4对紧密连接的调控过程，进一步揭示Robo4调控视网膜内皮细胞通透性的机制。结果显示：干涉Robo4后磷酸化cofilin（phosphorylatedcofilin, p-cofilin）和LIMK1的蛋白表达明显增加，为了进一步证实该信号途径，通过构建LIMK1siRNA、共转染Robo4siRNA和LIMK1siRNA后发现， p-cofilin的表达随之降低，同时伴随紧密连接蛋白的表达增加和应力纤维的减少，并且视网膜内皮细胞的通透性也得到恢复，说明LIMK1-cofilin信号途径参与了Robo4对内皮细胞通透性的调控过程。综上所述，Robo4在内皮细胞通透性方面起到重要作用，靶向干涉Robo4的表达增加了体外培养的单层视网膜内皮细胞通透性，，Robo4通过改变紧密连接从而对内皮细胞通透性进行调节，紧密连接相关蛋白occludin和ZO-1的表达下调和细胞骨架肌动蛋白F-actin的重排是导致内皮细胞通透性增加的直接原因，LIMK1-cofilin信号途径参与了Robo4对内皮细胞通透性的调控过程。
[Abstract]:Blood - retinal barrier is a kind of structure with restrictive permeability between blood and retina . The blood - retinal barrier includes inner barrier and outer barrier . Blood - retinal barrier consists of inner barrier and outer barrier . Blood - retinal barrier consists of inner barrier and outer barrier . Vascular endothelial cell is one of the main pathological changes in diabetic retinopathy , retinopathy of premature infants and Coats disease .

The increase in vascular endothelial permeability may have two mechanisms , one is the intracellular pathway and the other is the pathway pathway . It is found that the recombination and redistribution of the endothelial cells , especially actin , is the main pathological basis for the contraction of endothelial cells and the increase of vascular permeability .

Robo4 is the fourth member of Robo family and is a conserved transmembrane receptor protein . Robo4 , which is a fourth member of Robo family , is a conservative transmembrane receptor protein . Robo4 is a conservative transmembrane receptor protein . Robo4 is a conserved transmembrane receptor protein . Robo4 can block vascular endothelial growth factor ( VEGF ) downstream signaling pathway and participate in the regulation of endothelial cell migration . Recently , the literature report Robo4 can block vascular endothelial growth factor ( VEGF ) permeability , maintain the stability of mature vascular system , but the exact mechanism of Robo4 in retinal endothelial cell permeability has not been reported .

By observing the effects of Robo4 on the actin of closely linked protein and cytoskeletal actin , the mechanism of Robo4 in retinal endothelial cell permeability was discussed . The possible mechanism was discussed . First , the expression of Robo4 protein was detected in the course of increased permeability of endothelial cells induced by advanced advanced end product ( AGE ) .
The expression of Robo4 protein decreased with the increase of AGE action time , which showed that Robo4 protein could play a role in the regulation of permeability of retinal endothelial cells .
In order to further confirm the effect of Robo4 on retinal permeability , Robo4 siRNA specific silencing Robo4 expression was used to evaluate the change of monolayer permeability of retinal vascular endothelial cells by measuring the fluorescence emission intensity of dextran . The results showed that the inhibition of Robo4 expression in retinal vascular endothelial cells could increase the permeability of endothelial cells and destroy the barrier function of endothelial cells .

In the second part of the study , the expression of Robo4 protein in the endothelial cells of retinal vascular endothelial cells was inhibited by western blot .
At the same time , the cell skeleton actin F - actin in the cells was stained by rhodamine - ghost cyclopeptide , and there appeared a large number of dispersed stress fibers in the cytoplasm , while the peripheral dense bundles distributed around the periphery of the cells decreased significantly , which indicated that the monolayer permeability of the targeted knock - out Robo4 gene was increased , and the opening of the close connection could be closely related to the expression of the closely linked proteins , the expression of ZO - 1 and the recombination and redistribution of the cytoskeletal actin , and the mechanism of Robo4 regulating the permeability of endothelial cells was preliminarily clarified .

In the third part , we found that the expression of phosphorylation cofilin ( LIMK ) - cofilin ( ADF / cofilin ) and LIMK1 increased significantly in the third part . The results showed that the expression of phosphorylation cofilin ( LIMK ) - cofilin ( ADF / cofilin ) and LIMK1 increased significantly in the third part . The results showed that the expression of phosphorylation cofilin ( LIMK ) - cofilin and LIMK1 in Robo4 decreased , while the permeability of the retinal endothelial cells was restored . The expression of LIMK1 - cofilin signal was involved in the regulation of the permeability of Robo4 to endothelial cells .

In conclusion , Robo4 plays an important role in the permeability of endothelial cells , and the expression of targeted interference Robo4 increases the permeability of monolayer retinal endothelial cells cultured in vitro . Robo4 regulates the permeability of endothelial cells by changing the tight junctions , and the downregulation of the associated protein kinase din and ZO - 1 and the rearrangement of actin F - actin are the direct causes of increased permeability of endothelial cells .
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R774.1

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