神经轴突导向因子Slit2和受体Robo1、Robo4调控角膜血管新生

发布时间：2018-08-04 16:49

【摘要】： 背景与目的：角膜病是世界三大致盲眼病之一,中国公民因单眼和双眼角膜病致盲的盲人约四百万,占眼科致盲眼病的第2位。新生血管是角膜病致盲的主要原因,但其机制至今尚未完全清楚,目前亦无理想治疗方法,因此探索角膜新生血管形成机制及其防治措施具有重要的理论价值和实际意义。为了从全新的角度阐明角膜新生血管的分子机制,我们关注到与血管具有相似结构的系统——神经系统。在生物体内,神经和血管不但距离很近,而且彼此存在很多的关联,具有解剖的相似性和功能的平行性。鉴于神经和血管行为的相似性,两个系统可能共享相同的信号分子。目前已证实四个神经轴突导向因子家族同时调节血管发育,即Ephrins，Semaphorins, Netrins和Slits。我们将目标定位于神经系统中仅有单向调控作用的Slit及其受体Roundabout(Robo)家族。本研究将探讨神经轴突导向因子Slit2和Robo受体是否参与了角膜新生血管形成。方法：角膜囊袋法诱导大鼠角膜新生血管模型,实时荧光定量PCR测定Slit2和Robo 1-4在正常角膜与新生血管角膜的差异表达,免疫组化法和原位杂交法定位。体外培养原代的脐静脉内皮细胞,RT-PCR测定细胞Robo 1-4受体的表达。制备重组的人Slit2蛋白,测定其对血管内皮细胞迁移的影响。结果：大鼠新生血管角膜的Slit2表达量仅为正常角膜的55%,Robo1和Robo4受体表达量分别为正常角膜的2.7倍和1.77倍,差异呈显著性(P0.05)。Slit2、Robo1和Robo4受体均表达于正常角膜上皮层的全层。随着新生血管区角膜基质中血管内皮细胞侵入的增加,上皮层表达Slit2. Robo1和Robo4减少,而基质层血管内皮细胞表达Robo1和Robo4增多。原代的脐静脉内皮细胞仅表达Robo1和Robo4受体,且重组的人Slit2能抑制脐静脉内皮细胞的移行(P＜0.05)。结论：本研究首次探讨了神经轴突导向因子Slit2与角膜新生血管的关系。研究发现：Slit2通过Robo1、Robo4受体介导抑制角膜病理性新生血管过程,其可能成为角膜新生血管治疗的新靶点。
[Abstract]:Background & objective: corneal disease is one of the three major blinding eye diseases in the world. About 4 million blind people in China are blind due to monocular and biocular keratopathy, accounting for the second place in ophthalmic blinding ophthalmopathy. Neovascularization is the main cause of blindness caused by keratopathy, but its mechanism has not been fully understood and there is no ideal treatment at present. Therefore, exploring the mechanism of corneal neovascularization and its prevention and treatment has important theoretical value and practical significance. In order to elucidate the molecular mechanism of corneal neovascularization from a new perspective, we focus on the system-nervous system with similar structure to blood vessels. In vivo, nerves and blood vessels are not only close to each other, but also have many relations with each other, and have anatomic similarities and parallel functions. Given the similarity of neural and vascular behavior, the two systems may share the same signaling molecules. At present, it has been proved that four neuroaxon guiding factor families regulate angiogenesis at the same time, namely, Ephrinschus Semaphorinss, Netrins and Slits. We target Slit and its receptor Roundabout (Robo) family, which have only unidirectional regulation in the nervous system. This study will investigate whether Slit2 and Robo receptors are involved in corneal neovascularization. Methods: the rat corneal neovascularization model was induced by the capsule method. The differential expression of Slit2 and Robo 1-4 in the normal cornea and neovascularization cornea was detected by real-time fluorescence quantitative PCR, and the localization was performed by immunohistochemistry and in situ hybridization. The expression of Robo 1-4 receptor was detected by RT-PCR in primary umbilical vein endothelial cells cultured in vitro. The recombinant human Slit2 protein was prepared and its effect on the migration of vascular endothelial cells was measured. Results: the expression of Slit2 in neovascularization cornea was only 2.7-fold and 1.77 times as much as that of normal cornea (P0.05). The expression of Slit2Robo1 and Robo4 receptors were all expressed in the whole layer of normal corneal epithelium. With the increase of vascular endothelial cell invasion in corneal stroma of neovascularization area, Slit2 was expressed in the epithelium. Robo1 and Robo4 decreased, while Robo1 and Robo4 increased in stromal vascular endothelial cells. The primary umbilical vein endothelial cells expressed only Robo1 and Robo4 receptors, and recombinant human Slit2 could inhibit the migration of umbilical vein endothelial cells (P < 0. 05). Conclusion: the relationship between Slit2 and corneal neovascularization was studied for the first time. It has been found that Slit2 inhibits pathological corneal neovascularization via Robo1 and Robo4 receptor, which may be a new target for corneal neovascularization therapy.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R772.21

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