【摘要】： 背景與目的：角膜病是世界三大致盲眼病之一,中國(guó)公民因單眼和雙眼角膜病致盲的盲人約四百萬(wàn),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,但其機(jī)制至今尚未完全清楚,目前亦無(wú)理想治療方法,因此探索角膜新生血管形成機(jī)制及其防治措施具有重要的理論價(jià)值和實(shí)際意義。為了從全新的角度闡明角膜新生血管的分子機(jī)制,我們關(guān)注到與血管具有相似結(jié)構(gòu)的系統(tǒng)——神經(jīng)系統(tǒng)。在生物體內(nèi),神經(jīng)和血管不但距離很近,而且彼此存在很多的關(guān)聯(lián),具有解剖的相似性和功能的平行性。鑒于神經(jīng)和血管行為的相似性,兩個(gè)系統(tǒng)可能共享相同的信號(hào)分子。目前已證實(shí)四個(gè)神經(jīng)軸突導(dǎo)向因子家族同時(shí)調(diào)節(jié)血管發(fā)育,即Ephrins，Semaphorins, Netrins和Slits。我們將目標(biāo)定位于神經(jīng)系統(tǒng)中僅有單向調(diào)控作用的Slit及其受體Roundabout(Robo)家族。本研究將探討神經(jīng)軸突導(dǎo)向因子Slit2和Robo受體是否參與了角膜新生血管形成。方法：角膜囊袋法誘導(dǎo)大鼠角膜新生血管模型,實(shí)時(shí)熒光定量PCR測(cè)定Slit2和Robo 1-4在正常角膜與新生血管角膜的差異表達(dá),免疫組化法和原位雜交法定位。體外培養(yǎng)原代的臍靜脈內(nèi)皮細(xì)胞,RT-PCR測(cè)定細(xì)胞Robo 1-4受體的表達(dá)。制備重組的人Slit2蛋白,測(cè)定其對(duì)血管內(nèi)皮細(xì)胞遷移的影響。結(jié)果：大鼠新生血管角膜的Slit2表達(dá)量?jī)H為正常角膜的55%,Robo1和Robo4受體表達(dá)量分別為正常角膜的2.7倍和1.77倍,差異呈顯著性(P0.05)。Slit2、Robo1和Robo4受體均表達(dá)于正常角膜上皮層的全層。隨著新生血管區(qū)角膜基質(zhì)中血管內(nèi)皮細(xì)胞侵入的增加,上皮層表達(dá)Slit2. Robo1和Robo4減少,而基質(zhì)層血管內(nèi)皮細(xì)胞表達(dá)Robo1和Robo4增多。原代的臍靜脈內(nèi)皮細(xì)胞僅表達(dá)Robo1和Robo4受體,且重組的人Slit2能抑制臍靜脈內(nèi)皮細(xì)胞的移行(P＜0.05)。結(jié)論：本研究首次探討了神經(jīng)軸突導(dǎo)向因子Slit2與角膜新生血管的關(guān)系。研究發(fā)現(xiàn)：Slit2通過(guò)Robo1、Robo4受體介導(dǎo)抑制角膜病理性新生血管過(guò)程,其可能成為角膜新生血管治療的新靶點(diǎn)。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R772.21

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本文編號(hào)：2164497