VEGF介导白细胞瘀滞诱发可逆性视网膜血管闭塞及其机制研究

发布时间：2018-03-08 07:55

本文选题：VEGF　切入点：白细胞瘀滞　出处：《天津医科大学》2017年博士论文　论文类型：学位论文

【摘要】：背景:糖尿病视网膜病变(diabetic retinopathy,DR)中重要血管性改变—视网膜血管闭塞与视网膜静脉阻塞(retinal vein occlusion,RVO)继发的视网膜无灌注发生及缺血性视网膜病变成为成年人致盲重要原因。多项临床试验观察VEGF中和蛋白治疗糖尿病性黄斑水肿(diabetic macular edema,DME)与继发于RVO的黄斑水肿发现,抑制视网膜VEGF不仅能显著改善ME,而且能促进部分已闭塞的血管再通,阻止血管闭塞进展、微血管瘤的发生,提示VEGF可能在视网膜血管闭塞的发生中作用显著。而糖尿病诱发白细胞瘀滞参与视网膜微血管阻塞发生,因此本课题就VEGF是否可通过诱发白细胞瘀滞促进视网膜血管可逆性闭塞及其作用机制尚需进一步探讨。目的:(1)探讨VEGF对视网膜血管内白细胞瘀滞诱导作用;(2)观察VEGF诱发白细胞瘀滞是否诱发可逆性视网膜血管闭塞及视网膜缺氧发生;(3)探讨阻断VEGF是否能够逆转视网膜血管闭塞;(4)探讨VEGF诱发白细胞瘀滞的作用机制。方法:(1)C57BL/6鼠玻璃体腔注射不同浓度人VEGF(human VEGF,hVEGF)(0、50、100、200、500、1000ng/μl)后,予rhodamine或者FITC标记刀豆素蛋白(conconavalin,ConA)检测视网膜血管内白细胞黏附,并于荧光显微镜下计数。(2)Tet/opsin/VEGF双转基因鼠予四环素诱导视网膜光感受细胞短期内过表达较高量hVEGF;Rhodopsin/VEGF转基因鼠于出生后7天光感受细胞慢性持续性表达低浓度hVEGF,予rhodamine标记ConA标记黏附白细胞并计数。(3)荧光素钠血管造影(sodium fluorescein angiography,FA)后眼底照相观察hVEGF注射、Tet/opsin/VEGF及Rhodopsin/VEGF鼠视网膜血供情况,FITC-ConA灌注后行视网膜hypoxyprobe染色观察缺氧情况;(4)Tet/opsin/VEGF及Rho/VEGF鼠通过停止四环素诱导关闭VEGF表达或应用VEGF trap蛋白aflibercept后ConA灌注观察白细胞黏附,FA观察视网膜灌注恢复情况。(5)体外VEGF刺激人视网膜微血管内皮细胞(human retinal microvascular endothelial cells,HREC)后实时荧光定量聚合酶链反应(Quantitative Real Time-Polymerase Chain Reaction,qRT-PCR)检测黏附分子:细胞间粘附分子1(intercellular cell adhesion molecule-1,ICAM-1)、血管细胞粘附分子1(vascular cell adhesion molecule-1,VCAM-1),选择素(E-selectin,P-selectin),及NF-κB转录活性;体内qRT-PCR检测视网膜上述黏附分子及其配体黏附素表达,Western Blot、免疫荧光染色(immunofluorescence assay,IF)检测视网膜VCAM-1的分布及表达;WB视网膜下转染NF-κB(p65)reportor-luciferase载体检测视网膜核NF-κB(p65)的表达及其转录活性。FITC-ConA灌注后视网膜铺片F4/80、Ly6G免疫荧光检测黏附白细胞亚型。(6)尾静脉注射抗VCAM-1抗体及抗VEGFR1抗体后观察白细胞黏附改变。结果:(1)玻璃体腔注射50ng以上VEGF 24小时后诱发视网膜血管大量白细胞瘀滞呈剂量、时间依赖性表现为低剂量VEGF刺激白细胞黏附主要位于分支血管内;大剂量VEGF刺激白细胞大量瘀滞于扩张静脉及下游血管内。(2)Tet/opsin/VEGF转基因鼠视网膜VEGF表达随时间增加,血管白细胞瘀滞数目相应增加,且单个白细胞或者呈团块状聚集阻塞视网膜血管管腔;四环素诱导后2天、3天FA观察到视网膜静脉明显扩张、大量视网膜渗漏发生、及明显后极部血管闭塞、无灌注发生;且白细胞黏附处视网膜组织hypoxyprobe染色阳性提示缺氧发生;Rho/VEGF鼠于出生后20天开始检测到视网膜白细胞瘀滞发生于7月达到高峰并维持到15月,且黏附主要发生在分支血管、血管分叉处,可见单个细胞阻塞血管管腔;FA可见7月、12月鼠视网膜静脉扩张不明显,血管排布紊乱,后极部多处小面积无灌注发生,部分分布于视网膜下新生血管周围。(3)Tet/opsin/VEGF双转基因小鼠四环素诱导后3天FA检查可见视网膜后极部血管闭塞、小片状无灌注区域,而停止诱导14天后继续FA造影可见已闭塞血管恢复灌注,且ConA标记的黏附白细胞数目较3天时明显减少;应用aflibercept后,部分已闭塞的Rho/VEGF视网膜血管重新开放,血供恢复,同时周围新生血管也明显减少;而Tet/opsin/VEGF玻璃体腔注射aflibercept后白细胞黏附较对照组明显下降,FA可见视网膜血管形态基本正常、无明显渗漏及视网膜无灌注发生。(4)体外VEGF刺激HRECs4小时后,qPCR检测到VCAM-1、ICAM-1、E-selectin mRNA表达增加,其他黏附分子无明显改变;NF-κB(p65)活性实验检测其转录活性较PBS刺激组明显升高。(5)视网膜qPCR结果发现VEGF注射后24小时、Tet/opsin/VEGF四环素诱导后3天VCAM-1 mRNA水平表达升高,其他黏附分子无明显变化;同时IF发现到VEGF注射后大量VCAM-1表达于表层及内核层视网膜血管;WB检测到Tet/opsin/VEGF鼠高表达VEGF后3天视网膜胞浆蛋白中VCAM-1表达明显升高且阻断VCAM-1后视网膜ConA标记黏附白细胞较IgG组明显减少,同时WB也检测核蛋白中NF-κB于VEGF表达1天后开始升高并维持提示NF-κB被激活;NF-κB(p65)reportor-luciferase检测发现VEGF注射后luciferase活性较PBS注射组明显升高,提示VEGF启动NF-κB转录活性表达,并启动下游因子VCAM-1表达参与白细胞瘀滞发生。(6)视网膜铺片IF检测到部分ConA标记细胞F4/80阳性提示单核细胞VEGF诱导参与白细胞黏附、少部分表现为Ly6G阳性提示仍有少量中性粒细胞也参与该过程;阻断VEGFR1后,VEGF诱发的视网膜血管内白细胞瘀滞明显下降。结论:1)本研究阐明了视网膜局部VEGF高表达部分能够通过VEGFR1招募白细胞迁移到视网膜血管网,通过激活核转录因子NF-κB启动下游因子VCAM-1表达促进白细胞(单核细胞为主)黏附于视网膜血管内皮细胞,参与血管阻塞,且部分阻塞为可逆性。2)本研究也充分解释临床观察到的缺血性视网膜病变患者眼内注射VEGF中和蛋白治疗后出现的已经无灌注视网膜血管再灌注及视网膜无灌注进展的减少现象。3)我们研究也表明包括RVO、DR及其他缺血性视网膜病变的病因、病程尽管不同,但是他们均可认为是一类相似疾病即VEGF是加重疾病急剧进展的重要驱动因子。4)并初步探讨一种潜在单独持续抑制VEGF或联合VEGFR1、VCAM-1可能阻止早期RVO或PDR视网膜血管阻塞及进行性视网膜缺血发生的方法。
[Abstract]:Background: diabetic retinopathy (diabetic, retinopathy, DR) in the vascular change of retinal vascular occlusion and retinal vein occlusion (retinal vein occlusion, RVO) secondary to retinal non perfusion and ischemic retinopathy as adults to cause blindness. A number of clinical trials on VEGF and protein in the treatment of diabetic macular edema (diabetic macular edema DME), and macular edema secondary to RVO, inhibition of retinal VEGF can not only improve the ME, but also can promote the recanalization of occluded part, prevent blood tube occlusion in micro vascular tumors, suggesting that VEGF may be in retinal vascular occlusion has a significant role. The diabetes induced leukocyte stasis in retinal microvascular obstruction, therefore the issue of whether VEGF can promote blood stasis of white blood cells in retinal blood stagnation reversible closed tube Plug and its mechanism still need to be further explored. Objective: (1) to investigate the induction effect of VEGF on retinal blood white cells in the blood stasis; (2) to observe the VEGF induced leukocyte stasis is induced reversible retinal vascular occlusion and retinal hypoxia; (3) to investigate whether blocking VEGF could reverse (retinal vascular occlusion; 4) to investigate the mechanism of VEGF induced leukocyte stasis. Methods: (1) after intravitreal injection of different concentrations of C57BL/6 (human VEGF, VEGF hVEGF) (0,501002005001000ng/ L), to rhodamine or FITC labeled concanavalin protein (conconavalin, ConA) detection of retinal intravascular leukocyte adhesion, and count in the fluorescence microscope. (2) Tet/opsin/VEGF double transgenic mice with tetracycline induced photoreceptor cells within a short period of over expression of higher levels of hVEGF; Rhodopsin/VEGF transgenic mice at postnatal 7 day feeling expression of chronic persistent Low concentration of hVEGF, with rhodamine labeled ConA labeled leukocyte adhesion and counting. (3) fluorescein angiography (sodium fluorescein angiography, FA) after hVEGF injection for observation of fundus photography, retina blood Tet/opsin/VEGF and Rhodopsin/VEGF rats, FITC-ConA after reperfusion for retinal hypoxia were observed by hypoxyprobe staining; (4) Tet/opsin/VEGF and Rho/VEGF rats by stopping observation of leukocyte adhesion induced by tetracycline ConA perfusion closed VEGF expression or application of VEGF trap protein Aflibercept, FA to observe the retinal perfusion recovery. (5) VEGF in vitro stimulation of human retinal microvascular endothelial cells (human retinal microvascular endothelial cells, HREC) by real-time fluorescence quantitative polymerase chain reaction (Quantitative Real Time-Polymerase Chain Reaction, qRT-PCR) detection adhesion molecule: intercellular adhesion molecule 1 (intercellular cell adhesion molecule-1, IC AM-1), vascular cell adhesion molecule 1 (vascular cell adhesion molecule-1, VCAM-1), P-selectin (E-selectin, P-selectin), and NF- kappa B transcription activity; qRT-PCR in the detection of retinal adhesion molecules and their ligands adhesin expression, Western Blot, immunofluorescence staining (immunofluorescence assay IF) to detect the expression and distribution of VCAM-1 in retina WB; subretinal transfection of NF- kappa B (p65) reportor-luciferase carrier detection of retinal nuclear NF- kappa B (p65).FITC-ConA expression and transcription activity of retinal flatmount after reperfusion F4/80, Ly6G immunofluorescence adhesion of leukocyte subpopulations. (6) intravenous injection of anti VCAM-1 antibody and anti VEGFR1 antibody were observed after leukocyte adhesion changes results: (1) intravitreal injection of 50NG VEGF more than 24 hours after a large number of white blood cells induced by retinal vascular stasis in a dose, time-dependent performance for low dose VEGF stimulated leukocyte adhesion and To be located in the branch vessels; high dose VEGF stimulated a large number of white blood cells in venous stasis and expansion of downstream vessels. (2) retinal VEGF transgenic mice Tet/opsin/VEGF expression increased with time, the number of white blood cells and blood stasis increase, single white blood cells or in clumps aggregation of retinal vascular lumen obstruction; 2 days after induction of tetracycline 3 days, FA was observed in the occurrence of a large number of dilated retinal vein, retinal leakage, and obviously the posterior pole of the vascular occlusion, no perfusion occurred; and retinal leukocyte adhesion hypoxyprobe positive staining suggested that hypoxia occurred; Rho/VEGF rats in 20 days after birth to detect retinal leukocyte stasis occurred in July and reached the peak in 15 months, and the adhesion occurs mainly in vascular branches, vascular bifurcation, visible single cells blocking the lumen of blood vessels; FA visible in July, December in retinal vein dilation of blood is not obvious. Tube arrangement disorder, the posterior pole of the multiple small area without perfusion, located on subretinal neovascularization around. (3) Tet/opsin/VEGF double transgenic mice induced by tetracycline in 3 days after the FA examination showed posterior pole retinal vascular occlusion, patchy non perfusion area, stop induced 14 days later to continue FA angiography revealed occlusion vascular perfusion recovery, and the number of leukocyte adhesion to ConA markers compared with 3 days reduced; after the application of Aflibercept, has been part of Rho/VEGF occlusion of the retinal vessels reopened, restore blood supply, but also significantly reduce neovascularization; while Tet/opsin/VEGF intravitreal injection of Aflibercept after leukocyte adhesion was lower than the control group, visible form the retinal FA is basically normal, no obvious leakage and retinal non perfusion. (4) HRECs4 hours after VEGF stimulation in vitro, qPCR detected VCAM-1, ICAM-1, E-selectin increased the expression of mRNA Plus, other adhesion molecules had no obvious change; NF- kappa B (p65) activity test of its transcriptional activity than PBS stimulation group increased significantly. (5) retinal qPCR results showed that 24 hours after VEGF injection, 3 days VCAM-1 mRNA Tet/opsin/VEGF induced by tetracycline after elevated expression of other adhesion molecules has no obvious change; at the same time to find IF after the injection of VEGF, a high expression of VCAM-1 in the surface and inner retinal vessels; high expression of WB Tet/opsin/VEGF was detected in the retinas of VCAM-1 3 cytoplasmic protein expression was significantly increased and the blocking of VCAM-1 retinal ConA labeled leukocyte adhesion was significantly reduced compared to the IgG VEGF, and WB detection of nuclear protein NF- kappa B expression after 1 days increase and maintain that NF- kappa B was activated in VEGF; NF- kappa B (p65) reportor-luciferase detected VEGF after injection of luciferase activity compared with PBS injection group was significantly increased, suggesting that VEGF promoter NF- kappa B transcription activity table Up, and start the downstream factor of VCAM-1 expression is involved in leukocyte stasis. (6) retinal flatmount IF detected ConA labeled cells of F4/80 positive mononuclear cells induced by VEGF that involved in leukocyte adhesion, few showed positive Ly6G indicates that there are still a few neutrophils are involved in the process; the blocking of VEGFR1, retina the white cells in the VEGF induced vascular stasis decreased significantly. Conclusion: 1) the results of this study demonstrated the high expression of VEGF through VEGFR1 local retinal recruitment of leukocyte migration into retinal vascular network, through the activation of nuclear factor kappa B NF- and downstream factor VCAM-1 expression promote white blood cells (mononuclear cells) in retinal adhesion participate in vascular endothelial cells, vascular obstruction, and partial obstruction of reversible.2) this study also fully explain the ischemic retinopathy in patients with intraocular injection of VEGF and protein was observed after clinical treatment There has been no perfusion of retinal vascular reperfusion and retinal non perfusion progress reduce the phenomenon of.3) our study also showed that including RVO, DR and other causes of ischemic retinopathy, despite the different course, but they can be considered to be a kind of disease that is similar to VEGF with disease progression dramatically important driving factor.4) and preliminary to explore the potential of a single sustained suppression of VEGF or VCAM-1 combined with VEGFR1, RVO or PDR may prevent early retinal vascular occlusion and method of retinal ischemia occurred.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R774.1;R587.2

【相似文献】