环氧合酶-2及其抑制剂对角膜新生血管作用的研究

目的 观察大鼠角膜新生血管（CNV）形成过程中环氧合酶-2（COX-2）的表达,探讨COX-2抑制剂Celecoxib对CNV的作用.方法 采用免疫组织化学方法、RT-PCR法检测碱烧伤后COX-2和VEGF蛋白、mRNA在角膜中的分布,比较实验组和对照组COX-2与VEGF蛋白和mRNA的表达量,计算二者的相关性.结果 碱烧伤2 d,CNV芽形成,4 d出现成熟的新生血管腔.活化的COX-2、VEGF蛋白和mRNA的表达在碱烧伤24 h开始增加,4 d达峰,7 d开始回落,14 d仍有表达.COX-2主要表达于大鼠角膜损伤上皮及修复的上皮层、基质层中浸润的炎性细胞和新生血管内皮细胞,VEGF与COX-2的表达部位一致.实验Ⅱ组和Ⅲ组COX-2和VEGF蛋白、mRNA表达量与对照组的差异有统计学意义（P＜0.05）,实验Ⅰ组与对照组的差异无统计学意义（P＞0.05）.结论 COX-2在炎性CNV形成过程中表达上调,通过调控VEGF的表达而起重要作用.Celecoxib抑制COX-2的表达,抑制CNV形成.     

环氧合酶-2及其抑制剂对角膜新生血管作用的研究

目的:观察大鼠角膜新生血管(cornealneovasculariza-tion,CNV)形成过程中环氧合酶-2(COX-2)的表达情况及与CNV的关系,探讨COX-2抑制剂Celecoxib对CNV的抑制作用。方法:利用免疫组织化学方法、RT-PCR法检测碱烧伤后各个时期COX-2和VEGF蛋白、mRNA在角膜各层中的分布,并对其进行半定量。比较实验组和对照组各个时期COX-2和VEGF蛋白和mRNA表达量的差别,并对其进行统计学分析,了解COX-2与VEGF的相关性。结果:①活化的COX-2和VEGF蛋白、mRNA的表达在角膜新生血管的形成中均有一动态变化。②VEGF的表达区域和COX-2表达的部位高度一致。③实验II组和实验Ⅲ组COX-2和VEGF蛋白、mRNA表达量与对照组的差别有显著性(P<0.05),实验I组与对照组的差别无显著性(P>0.05)。实验组和对照组COX-2与VEGF的表达呈正相关。结论:①COX-2在炎症性角膜新生血管形成过程中表达上调,其调控VEGF的表达,在角膜新生血管的形成过程中起着重要作用。②Celecoxib能抑制COX-2的表达,有效的抑制角膜新生血管的形成。     

环氧化酶-2和血管内皮生长因子在激光诱导的大鼠脉络膜新生血管中表达的相关性

目的 探讨脉络膜新生血管(choroidal neovascularization,CNV)生成过程中环氧化酶2(cyclooxygenase-2,COX-2)和血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达及其意义.方法 532 nm激光诱导棕色挪威(Brown-Norway,BN)大鼠实验性CNV.光凝后7 d,采用免疫组织化学法检测COX-2和VEGF在CNV中的表达.采用Western blotting和RT-PCR方法检测光凝后1 d、3 d、7 d和14 d CNV中COX-2和VEGF蛋白与mRNA表达的变化.结果 CNV区域的血管内皮细胞和基质细胞均有COX-2和VEGF蛋白的表达,主要表达于胞浆中.COX-2蛋白及其mRNA的表达在光凝后3d时达高峰,VEGF蛋白及其mRNA的表达在光凝后7 d时达高峰.COX-2蛋白及其mRNA的表达早于VEGF蛋白及其mRNA的表达.结论 COX-2在CNV生成早期表达上调,且其表达先于VEGF的表达,提示COX-2可能通过调控VEGF的表达在CNV生成过程中发挥重要作用.     

玻璃体内注射塞来昔布对激光诱导大鼠脉络膜新生血管的抑制作用

目的观察选择性环氧化酶-2（COX-2）抑制剂塞来昔布对实验性大鼠脉络膜新生血管（CNV）的影响及其机制。方法雄性棕色挪威大鼠36只,以右眼为实验眼,激光光凝诱导实验性CNV模型。模型建立成功后,36只大鼠立即随机分为治疗组（n=18）和对照组（n=18）,分别玻璃体内注射10μL塞来昔布（1mg/mL）和10μL生理盐水。光凝后7d采用Westernblot（n=6）和RT-PCR（n=6）分别检测血管内皮生长因子（VEGF）蛋白及其mRNA以及COX-2mRNA的表达;光凝后14d采用苏木精-伊红染色（n=3）和脉络膜铺片（n=3）法分别检测CNV的厚度和面积。结果经塞来昔布治疗后,CNV的厚度和面积均明显减小（P=0.00）,VEGF蛋白及其mRNA表达均明显下降（P=0.02,P=0.03）,而COX-2mRNA无明显变化（P=0.59）。结论玻璃体内注射塞来昔布能够有效地抑制实验性CNV,其可能是通过抑制VEGF的表达而发挥作用的,为临床防治CNV相关性眼病提供新思路。     

诱生型一氧化氮合酶及环氧化酶2对氧诱导视网膜病变小鼠模型新生血管形成的影响

目的 探讨环氧化酶2（COX-2）和诱生型一氧化氮合酶(iNOS)对 氧诱导视网膜病变 （OIR）小鼠模型视网膜新生血管形成的影响及其可能的作用机制。方法 使用变化的氧浓 度诱导小鼠的视网膜新生血管形成。用免疫组织化学、实时定量聚合酶链反应以及Western blotting 检测COX-2、iNOS、基质金属蛋白酶-2（MMP-2）和血管内皮生长因子（VEGF）在视网膜新生血管形成过程中的表达，并研究COX-2或iNOS抑制对视网膜新生血管形成的影响以及在此过程中MMP-2及VEGF的表达变化。结果 COX-2或iNOS抑制显著下调了VEG F和MMP-2的表达并减少了视网膜新生血管的形成。iNOS抑制减少了COX-2的表达，而COX-2抑制也同样下调了iNOS 的表达.结论 在OIR小鼠模型的视网膜新生血管形成过程中存在COX-2 及iNOS的作用，它们对视网膜新生血管形成的影响可能通过调节VEGF和MMP-2的表达实现。     

环氧化酶-2抑制剂抑制大鼠角膜新生血管的研究

目的探讨环氧化酶-2（COX-2）抑制剂美洛昔康对角膜新生血管（CNV）的抑制作用及其作用机制。方法缝线法诱导大鼠CNV形成;将动物分5组：第1～3组分别为0．625、1．25、5．0mg／mL美洛昔康组,第4组为地塞米松组,第5组为阴性对照组。裂隙灯照相并计算CNV面积,RT—PCR法观察角膜VEGF和Ang-2 mRNA表达。结果第5组CNV面积明显高于其他各组（P〈0．05）。第5天、第8天时,第2组、4组CNV面积明显低于其余各组（P〈0．01）;第5天时,第4组CNV面积明显低于第2组（P〈0．05）。缝线后第8天、第23天,第2～4组Ang-2 mRNA、VEGF mRNA表达均低于第5组。结论COX-2抑制剂美洛昔康可明显抑制CNV的增生,可能通过减少VEGF、Ang-2的转录实现。     

小鼠角膜新生血管中ephrin-B2 mRNA的表达

在角膜新生血管（corneal neovascularization，CNV）形成过程中，多种细胞因子发挥着重要的调控作用。ephrin．B2为酪氨酸激酶家族成员之一，与其受体EphB4结合，参与血管发育和出芽血管的生成，在胚胎血管形成中起重要作用。另有研究表明，ephrin-B2在恶性肿瘤细胞及间质新生血管中起重要的调节作用。本实验旨在探讨ephrin-B2在病理性CNV形成中的作用及其表达程度与新生血管发生程度的关系。     

Bevacizumab治疗脉络膜新生血管疾病的现状

脉络膜新生血管（choroidal noevascularization，CNV）是引起多种眼底疾病视力障碍的主要原因。目前临床上大多应用的激光疗法和手术等治疗手段只能针对已生成的CNV，尚缺乏对CNV病因的针对性治疗。CNV的生成是一个多细胞因子参与的复杂过程，发生机制尚不清楚。已证实血管生长因子和抑制因子间的失衡是CNV发生的关键环节，而血管内皮生长因子（vascular endoclelial growth factor，VEGF）是重要的始动因素。近来发现的许多内源性和外源性血管抑制剂可从不同环节阻断CNV的发生发展，这使得针对CNV病因、尤其是针对VEGF的治疗成为研究的热点，以期从根本上有效地预防与治疗CNV。     

塞来昔布对实验性脉络膜新生血管的抑制作用

目的　观察选择性环氧化酶-2 (COX-2)抑制剂塞来昔布（celecoxib）对实验性脉络膜新生血管（CNV）的抑制作用。方法　鼠龄8~10周的健康雄性棕色挪威（BN）大鼠30只,随机分为空白对照组、实验对照组和塞来昔布治疗组,每组10只。塞来昔布治疗组采用灌胃法给药,剂量50 mg/kg,2次/d。给药后7 d,采用氪激光建立大鼠CNV模型,分别于激光光凝后3、7、14、21、30 d对实验对照组和塞来昔布治疗组所有大鼠行荧光素眼底血管造影(FFA)检查。激光光凝后21 d,每组随机处死5只大鼠,摘除眼球,制作空白对照组球后段组织切片、实验对照组和治疗组CNV膜切片。常规苏木精-伊红（HE）染色,计算实验对照组和塞来昔布治疗组的CNV膜相对厚度;采用免疫组织化学方法检测COX-2、血管内皮生长因子（VEGF）及基质金属蛋白酶-2（MMP-2)的表达。结果　激光光凝后21 d,塞来昔布治疗组 CNV发生率明显低于实验对照组（χ2=7.106 8,P=0.007 7）,CNV相对厚度较实验对照组明显减少（t=16.760 0,P=0.000 0）,COX-2、VEGF和MMP-2在CNV膜上的阳性表达均明显低于实验对照组（t=5.710 0,5.840 0,8.020 0;P=0.000 0）;空白对照组大鼠COX-2、VEGF和MMP-2在视网膜和脉络膜中的表达非常弱。结论　预防性服用塞来昔布能抑制激光诱导CNV的发生;通过抑制COX-2可减少CNV中VEGF和MMP-2的表达。     

环氧化酶-2抑制剂抑制眼部新生血管临床应用新进展

眼部新生血管一直是眼科界的难题之一,发病机制尚未完全清楚.目前,炎症与新生血管的关系成为了研究热点,其中环氧化酶_2(cyclooxygenase-2,COX-2)逐渐受到人们的重视.COX-2与新生血管密切相关,它通过诱导血管内皮生长因子等血管生长因子或其他途径采促进新生血管的生长.近年来,COX-2抑制剂对角膜新生血管、视网膜新生血管、脉络膜新生血管等抑制作用的研究取得进展,本文就COX-2的生物学特性及其抑制剂在抑制眼部新生血管方面的应用作一综述.     

COX-2 and its inhibitor Celecoxib in corneal neovascularization

To observe the expression of COX-2 in rat corneal neovascularization (CNV) and its relationship to CNV, and to explore the inhibition of Celecoxib, a COX-2 inhibitor, to CNV. · METHODS: The distribution and quantification of COX-2 and VEGF was detected by immunohistochemistry. Expression of COX-2 and VEGF mRNA was quantified by RT-PCR. The difference in protein and mRNA expressions of COX-2 and VEGF was analyzed to find the correlation between them. · RESULTS: Expression of activated COX-2 and VEGF protein and mRNA in CNV had a dynamic change. VEGF and COX-2 co-localized. Compared with the control group, expression of both protein, mRNA of COX-2 and VEGF in experimental group II and III had significant difference(P <0.05),indicating the correlation between COX-2 and VEGF, while that in experimental group I had no statistical difference (P >0.05). · CONCLUSION: COX-2 expression was up-regulated in inflammatory CNV. COX-2 modulates the expression of VEGF, playing a very important role in CNV. Celecoxib inhibit COX-2 expression so as to hold back the CNV.     

Recruitment of marrow-derived endothelial cells to experimental choroidal neovascularization by local expression of vascular endothelial growth factor

PURPOSE: The question of whether adult animals maintain a reservoir of endothelial progenitor cells (EPCs) in the bone marrow that is involved in neovascularization is under investigation. The following study was undertaken to examine the potential contribution of EPCs to the development of choroidal neovascularization (CNV) in adult mice and to examine the role of local expression of vascular endothelial growth factor (VEGF) in this process. METHODS: Lethally irradiated, adult female nude mice were engrafted with whole bone marrow isolated from male transgenic mice expressing LacZ driven by the endothelial specific Tie-2 promoter. Two months, following bone marrow reconstitution, confirmed by quantitative Taqman PCR, an E1-deleted adenoviral vector expressing vascular endothelial growth factor (165) (Ad.VEGF(165)) was injected subretinally to induce CNV, confirmed by collagen IV immunohistochemistry. Bone marrow-derived endothelial cells were detected using either X-gal staining or Y chromosome in situ hybridization. Y chromosome positive cells within the CNV were confirmed to be endothelial cells by lectin staining. RESULTS: Subretinal Ad.VEGF(165) was capable of inducing CNV. Four-week old lesions were found to contain LacZ expressing cells within the CNV in bone marrow transplanted animals but not in negative control animals. Eighteen percent of all Y chromosome positive cells within the CNV were found to be lectin positive while 27% of all endothelial cells within the CNV were Y chromosome positive. CONCLUSION: Engrafted bone marrow-derived EPCs were shown to differentiate into endothelial cells at the site of subretinal VEGF-induced CNV in mice. These results suggest that EPCs contribute to the formation of neovascularization and that subretinal expression of VEGF might play an important role in recruitment of these cells to the site of CNV.     

Topical nepafenac inhibits ocular neovascularization

PURPOSE: Topical nepafenac readily penetrates the cornea and is metabolized to amfenac, a potent cyclooxygenase (COX)-1 and COX-2 inhibitor. In this study, we tested the effect of topical nepafenac in three murine models of ocular neovascularization (NV). METHODS: A masked trial was performed to compare the topical effects of vehicle with one of several concentrations of nepafenac (0.01%, 0.03%, 0.1%, or 0.5%), 0.1% diclofenac, or 0.5% ketorolac tromethamine in mice with oxygen-induced ischemic retinopathy, mice with choroidal NV (CNV) due to laser-induced rupture of Bruch's membrane, or transgenic mice with increased expression of vascular endothelial growth factor (VEGF) in photoreceptors (rho/VEGF transgenic mice). RESULTS: Mice treated with 0.1% or 0.5% nepafenac had significantly less CNV and significant less ischemia-induced retinal NV than did vehicle-treated mice. Nepafenac also blunted the increase in VEGF mRNA in the retina induced by ischemia. In rho/VEGF transgenic mice, nepafenac failed to inhibit neovascularization. In additional studies, compared with vehicle-treated mice, mice treated with 0.1% or 0.03% nepafenac had significantly less CNV, whereas eyes treated with 0.1% diclofenac showed no significant difference. Mice treated with 0.5% ketorolac tromethamine for 14 days had high mortality, but when evaluated after 7 days of treatment showed no difference from mice treated with vehicle for 7 days. CONCLUSIONS: Topical nepafenac inhibits CNV and ischemia-induced retinal neovascularization by decreasing production of VEGF. The absence of effect in rho/VEGF transgenic mice is consistent with this mechanism. Topical nepafenac may provide an effective new treatment for ocular neovascularization. The excellent corneal penetration of nepafenac certainly plays an important role in this effect. It is possible that other antiangiogenic agents are also amenable to topical application after formulations are identified that maximize their corneal penetration. Because of the many advantages of the topical route of delivery, this is a possible topic for exploration.     

实验性脉络膜新生血管中环氧合酶-2、血管内皮生长因子和基质金属蛋白酶-2的表达

目的研究实验性脉络膜新生血管（CNV）大鼠中环氧合酶-2（COX-2）、血管内皮生长因子（VEGF）和基质金属蛋白酶-2（MMP-2）的表达,并进一步探讨其在CNV中的作用机制及相互关系。方法应用氪红激光光凝视网膜的方法诱导制作BN大鼠的CNV模型,分别选取并制作无任何干预的对照组和实验组光凝后第3、7、14、21、30天的＂最大CNV膜＂切片,采用免疫组织化学技术检测COX-2、VEGF和MMP-2在视网膜和CNV膜中的表达。应用HPIAS-1000型高清晰度彩色病理图文分析系统测定COX-2、VEGF和MMP-2阳性染色的平均灰度值。结果 COX-2、VEGF和MMP-2在正常视网膜和脉络膜中呈弱表达,视网膜光凝后,COX-2、VEGF和MMP-2在视网膜和CNV膜中的表达在7～14 d逐渐增强,21 d时三者的表达强度均达到高峰,之后略减弱。视网膜光凝后7、14、21、30 d,实验组COX-2、VEGF和MMP-2的免疫组织化学染色平均灰度值与对照组相比差异均有统计学意义（P〈0.05）。COX-2的表达强度与VEGF和MMP-2的变化均呈正相关（r=0.967,P=0.007;r=0.966,P=0.007）。结论激光诱导的实验性CNV中COX-2、VEGF及MMP-2的表达随着时间的延长呈动态变化,CNV局部炎症诱导的COX-2表达可能是VEGF和MMP-2的上游调节因子。     

Mecamylamine suppresses Basal and nicotine-stimulated choroidal neovascularization

PURPOSE: Nicotinic acetylcholine receptors (nAChR) are best known for their role in neurotransmission, but they have recently been demonstrated on vascular endothelial cells. Acetylcholine is their endogenous ligand, but they are also stimulated by nicotine. By stimulating nAChR, nicotine promotes tumor angiogenesis as well as atherosclerotic plaque neovascularization. In this study, the authors investigated the role of nAChR in the pathogenesis of choroidal neovascularization (CNV). METHODS: The effect of the nonselective nAChR antagonist mecamylamine was tested on human retinal and choroidal endothelial cells in vitro and in a murine model of CNV. RESULTS: Several nAChR isoforms were identified in retinal and choroidal microvascular endothelial cells, and the ability of these cells to form tubules when grown in growth factor-reduced basement membrane matrix and supplemented with VEGF was suppressed by the nAChR antagonist mecamylamine. Supplementation of the drinking water of mice with nicotine increased the size of CNV lesions at Bruch membrane rupture sites, an effect that was blocked by subcutaneous administration of mecamylamine (50 mg/kg/d) by an osmotic pump. In the absence of nicotine, CNV formation was suppressed by the infusion of 50 mg/kg/d mecamylamine or by topical application 0.1 or 1% mecamylamine to the cornea. CONCLUSIONS: These data suggest that endogenous activation of nAChR promotes CNV and that activation of nAChR by nicotine may contribute to the increased incidence of CNV seen in smokers with age-related macular degeneration (AMD). Topically administered mecamylamine could provide an appealing new treatment approach for CNV.     

脉络膜新生血管促进因子的研究进展

脉络膜新生血管(choroidal neovascularization,CNV)是危害视力的主要病变之一,为多种眼底疾病的最终结局,最终导致严重的视力丧失。脉络膜新生血管的产生与促进与血管内皮生长因子与促血管生成素有着密切的关系,我们就这两种细胞因子的来源与作用的研究进展进行综述。     

Stimulation of neovascularization by the anti-angiogenic factor PEDF

PURPOSE: Examine the effect of (pigment epithelium-derived growth factor; PEDF) on laser-induced choroidal neovascularization (CNV). METHODS: Adult C57Bl/6 mice were anesthetized and four laser spots were placed in each quadrant of the fundus with a krypton red laser (614 nm, 50 microm, 0.05 second, 200 mW). Animals were treated with various doses of PEDF administered with miniosmotic pumps implanted subcutaneously. Seven days after laser treatment, mice were perfused with 3% FITC high-molecular-weight dextran, the eyes enucleated, and neovascularization analyzed by confocal microscopy. Data were recorded as the volume of the neovascular complex. The effect of PEDF on endothelial cell migration, vascular tube formation in synthetic basement membrane, and VEGF production was also determined. RESULTS: Mice receiving a lower dose of PEDF (90 microg/mL) had significantly decreased areas of CNV. A high dose of PEDF (360 microg/mL) significantly increased CNV, whereas an intermediate dose (180 microg/mL) of PEDF had no effect. PEDF inhibited endothelial cell migration and vascular tube formation at lower doses (0.5-5 microg/mL). High doses of PEDF (25-50 microg/mL) stimulated endothelial cell migration, enhanced vascular tube formation in vitro, and stimulated VEGF production from endothelial cells. Neutralizing anti-VEGF antibody completely reversed the stimulatory effects of high doses of PEDF on CNV in vivo. CONCLUSIONS: PEDF demonstrates opposing effects on CNV and endothelial cell function. Whereas low doses are inhibitory, high doses can augment the development of the neovasculature. These results suggest that the effects of PEDF on neovascularization are more complex than originally believed and that caution should be exercised when PEDF therapies are considered.