esRAGE基因修饰OECs移植对糖尿病视网膜病变的治疗作用

发布时间：2018-03-29 18:39

本文选题：糖尿病视网膜病变　切入点：外生型内皮祖细胞　出处：《南昌大学》2014年硕士论文

【摘要】：背景及目的介绍：糖尿病视网膜病变（Diabetic retinopathy，DR）是糖尿病并发的主要微血管病变之一，已经成为全球成人致盲四大主因之一。而目前针对DR的治疗有药物、激光及抗VEGF治疗等，这种治疗只能起到防止病变继续恶化的作用，因此寻找DR的根治方法成为世界性的难题。DR是视网膜微血管内皮细胞损伤的一类疾病，，因此治疗DR修复微血管成为关键。近年发现外生型内皮祖细胞（Outgrowthendothelial cells，OECs）在微血管疾病中起到修复作用。AGEs-RAGE系统在糖尿病微血管病变中的重要作用已经得到明确，而内源分泌型RAGE（endogenoussecretory RAGE，esRAGE）作为RAGE中一种新型剪接异构体，能阻断AGEs介导的炎症反应。本文旨在探索esRAGE基因修饰OECs干预DR大鼠的治疗作用，观察该细胞对DR大鼠视网膜的影响以及对视网膜组织VEGF与TGF-β的表达的影响。方法：健康雄性SD大鼠50只，按照随机数字表随机分成4组，正常组8只，其余按1%链脲佐菌素（STZ）腹腔注射诱导造成DM模型（55mg/kg），正常对照组注射同等体积的柠檬酸缓冲液，饲养3个月后成DR模型，饲养过程中，死亡9只，再随机分为每组10只。通过眼底造影观察3个月、6个月DR的病变情况后，再进行分组，各组分别为正常对照组（CON），糖尿病视网膜病变组（DR），注射OECs组（OECs），注射esRAGE-OECs组（esRAGE-OECs），通过玻璃体注射等量体积等数量细胞溶液，于4周后行眼底造影观察眼底病变情况；麻醉、心脏灌注后摘取眼球，HE染色观察各组大鼠视网膜各层细胞病理改变；免疫组织化学技术检测VEGF和TGF-β在大鼠视网膜的表达情况等；结果：（1）3、6个月DR大鼠均现不同程度的视网膜眼底病变，前者出现明显渗漏，后者更加严重；（2）组织病理学提示：CON组：视网膜各层次结构规则整齐，形态正常；DR组：视网膜各层出现不同程度水肿，节细胞层微血管扩张充血，神经纤维层间距增大，垂直纤维断裂，内、外核层细胞结构疏松、排列紊乱，胞核增大，内核层相对较薄，神经节细胞层、内核层可见明显新生血管；注射OECs组：内丛状层、神经节细胞层水肿有减轻，内、外核层细胞排列较紧凑，仍然可见较多扩张血管；esRAGE-OECs组：视网膜组织水肿减轻，各层细胞较清晰规整，扩张血管有明显减少，并且周围有内皮细胞环绕；（3）免疫组织化学检测提示：与CON组比较，DR组、OECs组、esRAGE-OECs治疗组VEGF和TGF-β、的表达明显增多，差异有统计学意义（P＜0.05）；与OECs组和DM组相比较，esRAGE-OECs组VEGF、TGF-β的表达下降，差异有统计学意义（P＜0.05）。结论：在DR中，玻璃体注射esRAGE基因修饰的OECs能减少VEGF、TGF-β的表达，可能改善视网膜组织的供血，起到治疗DR的作用。
[Abstract]:Background and objectives:Diabetic retinopathy (DRR) is one of the major microvascular lesions in diabetes mellitus and has become one of the four major causes of blindness in adults worldwide.At present, there are drugs, laser and anti- VEGF therapy for Dr. This kind of treatment can only prevent the disease from getting worse.Therefore, finding a radical cure for Dr becomes a worldwide problem. Dr is a kind of disease of retinal microvascular endothelial cell injury, so the treatment of Dr repair microvascular becomes the key.In recent years, it has been found that exogenous endothelial progenitor cells (OECs) play an important role in the repair of microvascular diseases. The important role of AGEs-RAGE system in diabetic microangiopathy has been clarified, while endogenous secretory RAGE(endogenoussecretory RAGEG is a new splicing isomer in RAGE.It can block the inflammatory response mediated by AGEs.The aim of this study was to explore the therapeutic effect of esRAGE gene modified OECs on Dr rats, and to observe the effect of the cells on the retina of Dr rats and the expression of VEGF and TGF- 尾 in retina.Methods:Fifty male Sprague-Dawley rats were randomly divided into 4 groups: normal group (n = 8). The rest were induced by intraperitoneal injection of 1% streptozotocin (STZ) to induce DM model. The normal control group was injected with the same volume of citric acid buffer.The pathological changes of Dr were observed by fundus angiography for 3 months and 6 months, and then divided into groups.Each group was divided into normal control group, diabetic retinopathy group, OECs group and esRAGE-OECs group, respectively. After 4 weeks, fundus angiography was performed to observe the fundus lesion.The histopathological changes of retinal cells in each group were observed by HE staining after cardiac perfusion, and the expression of VEGF and TGF- 尾 in the retina of rats were detected by immunohistochemistry.Results:Different degrees of retinopathy were found in Dr rats at 6 months. The former showed obvious leakage, and the latter was more serious. Histopathology suggested that the structure of the retina was regular, and the structure of the retina was regular.In Dr group with normal morphology, edema appeared in various layers of retina, microvascular dilatation and hyperemia in ganglion cell layer, increase of nerve fiber layer spacing, rupture of vertical fiber, loose structure of inner and outer nuclear layer, disorder of arrangement and enlargement of nucleus.The nuclear layer was relatively thin, the ganglion cell layer and the nuclear layer were obviously neovascularization. In the OECs injection group, the edema of the ganglion cell layer was alleviated, and the cells in the inner and outer nuclear layer were arranged compactly.There were still more dilated vessels in esRAGE-OECs group: the edema of retinal tissue was alleviated, the cells in each layer were clear and regular, and the vasodilation was obviously decreased.The difference was statistically significant (P < 0.05).Conclusion:In Dr, vitreous injection of OECs modified by esRAGE gene can reduce the expression of VEGF TGF- 尾, and may improve the blood supply of retina and play a role in the treatment of Dr.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R587.2

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