2011-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/648699摘要：正常情況下人體眼睛視網膜的血管在出生後就不再生長，所有出生後的網膜新生血管視為一種不正常的病理變化，如在老人性黃斑部病變、糖尿病視網膜症、新生兒視網膜症、及視網膜靜脈血管阻塞等疾病都會造成嚴重的併發症，包括玻璃體視網膜出血，視網膜纖維化，進一步形成牽引性視網膜剝離。黃斑部出血及纖維增生等導致視力嚴重喪失。另外新生血管會在虹膜及前房隅角生成，造成新生血管性青光眼。這些都是視網膜新生血管併發症造成視力喪失重要的原因。在糖尿病增殖性視網膜病變病人其玻璃體中的VEGF 濃度明顯上升，同時VEGF 可引發內皮細胞增生。雖然抑制VEGF 可減少視網膜血管新生，但它仍不能完全抑制因缺氧或發炎引起之視網膜血管增生。因此，應有許多其它重要因子參與血管新生的致病機轉。CCN family 是一群結構相似的蛋白質家族，成員包括CTGF、CYR61、Nov、WISP-1、WISP-2、及WISP-3。Cysteine-rich 61 (CYR61)是第一個被clone 出來的CCN family。大部份的CCN family 有相似的分子結構，但有不同的細胞功能，包括：細胞分裂的調節，細胞趨化作用，細胞凋亡，細胞黏合、移行、血管新生等。本實驗室先前研究發現在增殖性糖尿病網膜病變的致病機轉中，CYR61扮演一個重要的角色。我們發現CYR61在缺氧的細胞模式及小鼠模式中可被引發，同時在糖尿病的大鼠模式中也發現其視網膜表現CYR61，增殖性糖尿病病患玻璃體中的CYR61濃度也較對照組高。我們也發現CYR61可誘發視網膜內皮細胞的趨化作用及血管管狀物形成。所以CYR61參與了增殖性糖尿病網膜病變的致病機轉。然而，CYR61在眼球引發血管新生作用，除了本身的作用外，是否另外又引發其他血管增生因子參與眼球血管增生作用及其作用機制及訊息傳導路徑，至今仍未研究透徹。許多的Chemokine參與了細胞的趨化作用及血管管狀物形成等血管新生作用，特別如MCP-1等，MCP-1除了趨化單核球外，它也和血管內皮細胞及腫瘤細胞移行作用有關，有促進血管新生的作用。COX-2可合成prostagladins，可增加VEGF的分泌，為一個重要的血管增生因子，其和腫瘤惡化及擴散有關，同時也是引起視網膜血管增生的重要因子。金屬蛋白酶(Matrix metalloprotease，MMP)在血管新生形成中，內皮細胞在間質中的移行及形成有著重要的關係，特別是MMP-2及MMP-9。在我們研究室的先期實驗發現CYR61可引發MCP-1及COX-2的表現。在CYR61可誘發MCP-1、COX-2及MMPs下，促進其血管新生的假設下，我們預定要進行為期三年之研究計劃，其主要目的有三：一、在CYR61的刺激下引發Chemokines特別是MCP-1及COX-2，和MMPs特別是MMP-2及MMP-9的表現，及其對視網膜血管內皮細胞趨化作用及管狀物形成的研究。二、利用高氧誘發小鼠視網膜血管增生及大鼠玻璃體內注射CYR61蛋白模式去研究MCP-1、COX-2及MMP-2、MMP-9的表現。三、利用integrin 拮抗劑、蛇毒血清中的disintegrin及各種訊息因子活化抑制劑去研究CYR61引發這此因子的訊息分子的表現，及調控機轉。我們希望進一步去證實CYR61 在血管新生作用上扮演的角色，藉以了解CYR61 在視網膜新生血管增生之致病機轉，並尋求新的可行治療之道。<br> Abstract: Angiogenesis, the growth and proliferation of new blood vessels, is animportant aspect of the vasculoproliferation found in tumor growth, wound repair,inflammatory states, and ischemic sequel in the ocular angiogenetic diseases. Diabetesmellitus, occlusion of the central retinal vein or prematurity with subsequent exposureto oxygen can all be associated with intraocular neovascularization, which may resultin vitreous hemorrhage, tractional retinal detachment, neovascularization glaucomaand blindness. A number of mediators involve the angiogenic process. These includebasic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I), vascularendothelial cell growth factor (VEGF),2-4 and CCN family.Cysteine-rich 61 (Cyr61) is the first cloned member of the CCN family,which comprises Cyr61/CCN1, connective tissue growth factor (CTGF/CCN2),nephroblastoma overexpressed (Nov/CCN3), Wisp-1/elm1 (CCN4), Wisp-2/rCop1(CCN5), and Wisp-3 (CCN6). In our previous studies, we had demonstrated thatCyr61 could be induced under hypoxia in vitro and in vivo models, expressed inretina of streptozocin-induced diabetic rats, significantly increased in the vitreouslevels of patients of proliferative diabetic retinopathy (PDR), and could inducedmigration and tube formation in retinal vascular endothelial cells. Therefore, Cyr61plays an important role in the pathogenesis of PDR.Several proinflammatory factors involve in the process of new vesselformation. Monocyte chemoattractant factor-I (MCP-1) and cyclooxygenase-2(COX-2) has been reported to mediate vascular endothelial cell adhesion, chemotaxis,capillary tube formation, and angiogenesis. Metalloproteinases (MMPs), especiallyMMP-2 and MMP-9, have been revealed to enhance cell migration and angiogenesis.In our preliminary study, we had found that CYR61 induced expression of MCP-1and COX-2 in cultured retinal vascular endothelial cells. However, the mechanisms ofthe Cyr61 induced angiogenesis were not well understood. In this three year project,our will divided our experiments into 3 parts. In the first year, we will investigate theexpression of chemokines and MMPs in the mRNA and protein levels under thestimulation of CYR61 in retinal vascular endothelial cells and evaluate theirangiogenic effects under CYR61 stimulation. In the second year, we will designmodels of oxygen-induced retinopathy of mice and intravitreal injection of CYR61protein to observe the expression of these proinflammatory factors and their roles inthe angiogenesis in the retina. In the third year, we will use disintegrin and otherinhibitors to investigate the signaling pathways including MAPK pathway, NF-κB,PI3K-Akt pathway, and ILK-β-catenin pathway. Our results might further understandthe mechanism of the angiogenic effect of CYR61 and found a new therapeuticstrategy for the treatment of ocular angiogenesis, such as PDR.血管增生因子Cyr61環氧合?(COX)-2增殖性糖尿病視網膜病變核蛋白 NF-κB血管新生作用