白细胞源性微粒在缺血性视网膜病变中作用机制的研究

Diabetic retinopathy and retinal vein occlusion are the top two vision-threating eye diseases for adults worldwide without clear mechanism clarified. As what we reported before, VEGF as the predominant factors can induce large amount of leukocyte expressed VEGFR1 recruitment and plugging the retinal vessels, which cause vascular occlusion and ischemia and hypoxia subsequently. However, the mechanism involved was not clear yet. Leukocyte-derived microparticles are hotshot due to their effect of proinflammation and procoagulation . Leukocyte-derived microparticles were increased in vitreous collected from diabetic retinopathy patients, and decreased with anti-VEGF treatment.Accordingly, we hypothesize that VEGF induce the release of microparticles via activation of VEGFR1, which promote coagulation, inflammation and endothelial injury, and then enhance the progression of retinal vascular occlusion. In this project, we would like to set up VEGF overexpressed animal model and peripheral blood leukocytes stimulated with VEGF, and perform flow cytometry to test leukocyte derived microparticles ;we will pursue their effect on the regulation of TF/TFI balance, expression of inflammatory factors and damage of endothelial cells, hitherto, cause retinal vascular occlusion. We will look into the mechanism of VEGF release leukocyte derived microparticles via VEGFR1 pathway. These results will promote better understanding of pathology and treatment of retinal ischemic retinopathy.

糖尿病视网膜病变与视网膜静脉阻塞是世界范围内诱发成年人视力丧失的两大首要缺血性视网膜病变。申请人前期工作显示VEGF能够诱发视网膜血管表达VEGFR1白细胞瘀滞参与血管闭塞及缺血,但黏附后白细胞参与视网膜血管闭塞的机制尚不十分明确。白细胞源性微粒有促炎促凝等生物活性而备受关注，申请人亦发现其在糖尿病视网膜病变患者玻璃体中表达升高，且抗VEGF应用后明显下降。在此基础上，本课题假设VEGF可激活VEGFR1释放白细胞源性微粒，通过促凝、扩大炎症反应及破坏血管内皮细胞参与视网膜血管闭塞发生。本项目将建立VEGF转基因动物模型并体外刺激外周血白细胞，检测白细胞源性微粒体表达；探究其对组织因子/组织因子抑制因子调节，炎症因子表达及诱发内皮细胞凋亡等，发挥促炎促凝及内皮细胞损伤作用，诱发视网膜血管闭塞；探究VEGF通过启动VEGFR1通路诱发白细胞源性微粒释放机制，为缺血性视网膜病变诊疗提供新思路。

糖尿病视网膜病变与视网膜静脉阻塞是世界范围内诱发成年人视力丧失的两大首要缺血性视网膜病变。VEGF能够诱发视网膜血管表达VEGFR1白细胞瘀滞参与血管闭塞及缺血,但黏附后白细胞参与视网膜血管闭塞的机制尚不十分明确。白细胞源性微粒有促炎促凝等生物活性在脑卒中、脑外伤等缺血性疾病中发挥有重要作用。在本项目中，我们建立了视网膜高表达VEGF动物模型，验证了视网膜高表达VEGF后视网膜白细胞源性微粒（LDMP）表达显著性升高，同时体外VEGF刺激鼠外周血白细胞后，其LDMP表达量明显升高，表明视网膜高表达VEGF可以促进LDMP表达；进一步检测LDMP表达的相关炎症因子IL-1β等表达增加，凝血因子（TF）表达增加，提示LDMP导致视网膜炎症及视网膜凝血功能异常。我们提取足量VEGF刺激LDMP，注射到正常小鼠体内后，可以观察到LDMP诱发的视网膜血管渗漏，视网膜血管闭塞及视网膜局部hypoxyprobe阳性表达；同时视网膜血管白细胞黏附增加，视网膜血管内皮细胞PI阳性表达增多，Ve-cadherin表达减少；LDMP与人视网膜血管内皮细胞（HREC）共培养后观察可见凋亡HREC增多，Bax等凋亡蛋白表达增加，而血管内皮屏障VE-cadherin表达减少；表明VEGF诱发的LDMP可以通过促炎促凝破坏血管内皮导致视网膜血管缺血缺氧。体外实验证实VEGF可以通过激活白细胞表面VEGFR1及下游ERK/p38信号通路破坏细胞骨架蛋白Actin表达，促进LDMP产生，阻断VEGFR1后，LDMP表达量明显下降。视网膜高表达VEGF小鼠及体外VEGF刺激时给予Lactadherin蛋白可以观察到VEGF诱发的白细胞源性微粒表达明显下降。VEGF可激活白细胞表面的VEGFR1诱发下游信号通路破坏细胞骨架蛋白Actin表达，促进白细胞源性微粒释放，诱发TF表达增加、破坏TF/TFI平衡加重凝血，促炎因子释放，及诱发内皮细胞破坏进而导致视网膜血管闭塞，视网膜缺血性病变发生，正反馈性促进VEGF表达增加，加重缺血性视网膜病变进展，Lactadherin蛋白阻断LDMP释放在一定程度上减轻缺血性视网膜病变。本研究结果为临床缺血性视网膜病变的发病机制提供新的理论基础，寻找新的作用靶点。基于这些研究结果，在本基金的支持下，共发表SCI论文3篇。

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