【作者】 张朝霞；

【导师】 王雨生；

【作者基本信息】 第四军医大学 ， 眼科学， 2009， 博士

【摘要】 研究背景脉络膜新生血管(choroidal neovascularization, CNV)是年龄相关性黄斑变性(age related macular degeneration, AMD)导致严重视力丧失最主要的原因;其发病是一多因素参与的复杂过程,但确切机制仍未阐明。血管生成和血管发生是参与新生血管形成的两种机制。一般来说,局部促血管生成因子参与血管生成,其主要细胞成份来源于已存在的毛细血管。然而,近年研究发现外周血骨髓来源的内皮祖细胞(endothelial precursor cells, EPCs)可能参与病理性新生血管形成,包括视网膜和脉络膜新生血管。作为CXC趋化蛋白超家族成员之一,基质细胞衍生因子-1(stromal cell derived factor-1, SDF-1)参与趋化骨髓来源细胞(bone marrow derived cells, BMCs)向靶组织募集,其受体CXCR4可表达于多种细胞,包括内皮细胞及EPCs。体内、外研究证明,SDF-1也是一种促血管生成因素。SDF-1或CXCR4基因缺陷小鼠中消化道大血管发育缺失。增殖性视网膜病变和CNV模型显示,SDF-1/CXCR4可诱导EPCs聚集于新生血管形成部位,而CXCR4拮抗剂可明显抑制包括缺血性视网膜新生血管和CNV在内的多种眼内新生血管病变。临床研究表明,正常老年人及AMD患者脉络膜中,视网膜色素上皮(retinal pigment epithelium, RPE)和脉络膜损伤处SDF-1和CXCR4高表达。组织缺血时,缺氧诱导因子-1α(hypoxia inducible factor-1α, HIF-1α)可上调SDF-1表达,介导EPCs向缺血组织聚集,而CNV形成过程中也存在HIF-1α表达增加。RPE细胞可持续表达SDF-1,并在缺氧时表达增加。然而,SDF-1在CNV发生中的作用机制尚未完全阐明。目的和内容探讨SDF-1在CNV形成过程中的作用,进一步阐明CNV的发生机制。在激光诱导的小鼠CNV模型中,观察HIF-1α和SDF-1表达;体外通过化学缺氧及运用HIF-1小干扰RNA(small interfering RNA, siRNA)观察SDF-1表达。观察C57BL/6J-GFP嵌合体小鼠CNV模型中SDF-1诱导骨髓来源的EPCs和内皮细胞(endothelial cells, ECs)在CNV局部的聚集;体外模拟在不同SDF-1表达水平及微环境下新生血管生成情况,观察SDF-1对BMCs分化的影响。方法1)利用532nm激光建立小鼠CNV模型,观察CNV局部HIF-1α和SDF-1的表达;利用体外培养RPE细胞,观察缺氧条件下,RPE细胞HIF-1α和SDF-1表达的情况;运用siRNA技术构建HIF-1α低表达的RPE细胞,观察缺氧状态下HIF-1α缺失对于RPE细胞SDF-1表达的影响;2)建立C57BL/6J-GFP嵌合体小鼠CNV模型,观察CNV局部SDF-1表达及BMCs表面CXCR4、CD34、CD31和c-kit表达;3)体外培养人原代BMCs和RF/6A细胞,通过三维共培养技术,比较不同SDF-1表达水平及不同微环境下,BMCs参与新生血管形成及发生分化的情况。结果免疫荧光染色显示CNV部位HIF-1α表达升高,于24h达峰值;随之SDF-1表达也升高,于3d达峰值,表达部位均位于近RPE层附近。缺氧诱导体外培养RPE细胞HIF-1α及SDF-1表达增高,于4小时达峰值;shHIF-1α抑制HIF-1α表达,同时SDF-1表达下调。脉络膜铺片及冰冻切片间接免疫荧光染色结果表明,在CNV形成极早期,GFP阳性的BMCs主要聚集于SDF-1高表达的光斑周围的RPE层附近,形态较均一,其中包含大量EPCs细胞;CNV形成过程中,大量BMCs细胞进入损伤内部,部分细胞胞体变狭长,分化为内皮细胞(CD31+)形成管腔样结构,此时损伤周边仍聚集大量的EPCs;随CNV形成,可见大量新生血管管腔样结构交织成网,大量BMCs细胞分化的内皮细胞胞参与其形成。CNV损伤局部聚集的BMCs均为CXCR4阳性,其中CD34阳性和/或c-kit阳性EPCs位于损伤周边部,而同来源的CD31阳性内皮细胞随CNV发展,渐伸入损伤核心区,参与新生血管形成。体外三维胶原纤维基质模型显示,SDF-1高表达组新生血管密集,单纯SDF-1升高即可诱导BMCs向内皮细胞分化,参与新生血管形成。结论在CNV形成过程中,缺氧诱导RPE层HIF-1α表达增高,上调SDF-1表达;SDF-1/CXCR4趋化BMCs聚集于CNV局部,并诱导部分BMCs向内皮细胞分化,参与CNV新生血管形成。更多还原

【Abstract】 Background Choroidal neovascularization (CNV) is a major cause of vision loss in patients with age related macular degeneration (AMD). The pathogenesis of CNV is clearly multifactorial, but not completely understood. Generally, neovascularization incorporates two forms of new blood vessel growth: angiogenesis and vasculogenesis. Angiogenesis were generally promoted by growth factors, and most cellular components of the new vessel complex (endothelial cells, smooth muscle cells, and pericytes) were derived from cells resident within adfacent pre-existing blood vessels. However, more and more studies in animal models suggest that circulating bone marrow-derived endothelial precursor cells (EPCs) may be participate in pathophysiological noevascularization, including retinal and choroidal neovascularization.As a member of the CXC chemokine subfamily, stromal cell derived factor 1 (SDF-1) is implicated for CXCR4-positive EPCs recruitment from the bone marrow to the target tissue. CXCR4, a seven transmembrane spanning G protein coupled receptor, is the only known receptor for SDF-1 and is expressed on various endothelial cells and EPCs. It has been reported that SDF-1 acts as an angiogenic agent in several in vivo and in vitro model systems. Mice deficient in either SDF-1 or CXCR4 had defective formation of large vessels supplying the gastrointestinal tract. In proliferative retinopathy and CNV model, SDF-1/CXCR4 may have a role in orienting EPC to reach sites of neovascularization. Besides, CXCR4 antagonists were effective supppressors of several types of ocular NV, such as ischemia-induced retinal NV and CNV. Clinically, in aged control and AMD choroid, SDF-1 and CXCR4 were most prominent in retinal pigment epithelial (RPE) cells and choroidal stroma.Some studies showed that SDF-1 was expressed in ischemic tissues under the control of HIF-1α. In models of wound healing and several models of tissue hypoxia, SDF-1 mediated homing of endothelial progenitor cells to blood vessel walls in the ischemic tissue. It was concluded that recruitment of CXCR4 positive progenitor cells to regenerating tissues were mediated by hypoxic gradients via HIF-1α-induced expression of SDF-1. HIF-1αinduced SDF-1 expression facilitated adhesion of progenitor cells to ischemic endothelium and induced their migration in transwell assays, suggesting a specific role in homing of circulating progenitor cells to ischemic vascular beds. In this study, we will investigate the potential role of hypoxia induced SDF-1 expression in formation of CNV.Objectives It would be helpful for our understanding of CNV to study the effect of SDF-1 on CNV formation. In this study, we investigated the expression of HIF-1αand SDF-1 in CNV models; then, we studied the SDF-1 expression in cultured RPE treated with CoCl2 and HIF-1siRNA. Then, we used C57BL/6J-GFP chimeric mice to investigate the SDF-1 induced bone marrow derived-EPCs to CNV. Finally, We established an in vitro 3-dimensional (3D) matrix system to study the neovascularization under different SDF-1 levels and microenviroments.Methods 1) The C57BL/6J mice underwent laser rupture of Bruch’s membrane to induce CNV and were killed at 1, 4, 8, 16, 24 hours and 1, 3, 7, 14, 28 days after laser injury. Immunofluorescence analysis were processed the expression of HIF-1αand SDF-1. Cultured human RPE cells were exposed to hypoxia (CoCl2, 0.2mM) for 0, 1, 2, 4, 8, 16, 24 and 48 hours, HIF-1αand SDF-1 was detected with Western Blot, RT-PCR and ELISA respetively. HIF-1αshRNA was used to knock down the HIF-1αgene in hRPE, the expression of HIF-1αand SDF-1 under hypoxia was detected. 2) Green fluorescent protein (GFP) chimeric mice were developed by transplanting bone marrow cells from GFP+/+ transgenic mice to adult C57BL/6J mice and established CNV model after 1 month when confirmed the successful bone marrow transplantation. The chimeric mice were killed at 1, 3, 7, 14 and 28 days after laser injury. The eyes were enucleated and frozen section. SDF-1, CXCR4, CD34, CD31 and c-kit were detected by immunofluorescence. The recruitment of BMCs to CNV is measured by choroidal flatmount. 3) 3D fibrin matrix system was prepared in vitro with human fibrinogen, thrombin and aprotinin. Cultured human BMCs (marked with DiO) and RF/6A cells were seed in the gel, cocultured with different conditional media. The tubular structure and BMCs participating in neovascularization were investigated with a phase contrast microscope. Gels were fixed in 4% buffered formalin and processed for paraffin embedding. The embedded matrixes were cut (5μm) perpendicularly to the surface of the matrix sheet. The expression of CD34 and CD31 on BMCs was examined by immunofluorescence.Results Immunofluorescence showed that the expression of HIF-1αand SDF-1 in CNV were near RPE layer, which were gradually higher and reached the peak at 24 hour and 3d respectively after laser injury. RT-PCR and Western Blot showed that the hypoxic stimulus induced the expression of HIF-1αand SDF-1 in cultured hRPE, and reached the peak at 4th hour, while shHIF-1αhad knockdown the expression of HIF-1αand down-regulated the SDF-1 level. Choroidal flatmount showed that at the early stage of CNV, BMCs gathered around RPE layer near the laser lesion; the GFP-labeled cells were morphologically green fluorescent round spots. During CNV formation, CXCR4 marked BMCs in CNV lesion were differentiated into endothelial cells (CD31+) and participated in vessel formation. When CNV came into being, there were amount of BMCs derived endothelial cells participating in the neovasculature network. Immunofluorescence showed that most BMCs gathered in CNV were CXCR4 positive cells, and some acquired phenotypic markers of EPCs (CD34 and c-kit) and ECs (CD31). In the RF/6A-BMCs-cocultured 3D fibrin matrix system, the DiO labled BMCs were particitated in the tubular networks. There were more CD31 positive BMCs (Dio labeled) in SDF-1 constitutive-expression, SDF-1 and hypoxia group.Conclusion During CNV formation, the hypoxia induced higher expression of HIF-1αin RPE layer, which up-regulated the SDF-1 expression. SDF-1 and its receptor CXCR4 were critical mediators for ischemia-specific recruitment to CNV. Also did SDF-1 can induce BMCs differentiate into endothelial cells to participate in the neovascularization.更多还原