中文摘要：

背景优化人视网膜血管内皮细胞的培养和鉴定方法在视网膜血管性疾病的研究中有重要作用，以往研究者已成功培养了人视网膜血管内皮细胞，但进一步简化其方法并达到获取量大而纯的细胞是基础研究和临床研究的基础。目的探索一种更为简便快速、收获量大且纯度高的视网膜血管内皮细胞的改良培养方法，并对目标细胞的抗原表达特点进行分析，比较眼科新生血管性疾病研究中常用的两种血管内皮细胞的标志性蛋白表达情况。方法利用正常人角膜移植供体眼球分离视网膜组织，采用质量分数2％胰蛋白酶和质量分数0．133％胶原酶I用二步消化法获取人视网膜血管内皮细胞，在传统培养方法中采用含质量分数10％胎牛血清的人血管内皮细胞培养液的基础上，添加内皮细胞生长因子一B（13-ECGF）和肝素钠，对分离的细胞进行体外培养，培养皿用纤维黏连蛋白（FN）包被以促进培养细胞的贴壁。观察收获的目标细胞的形态特征，采用活体显微镜进行形态学观察、常规组织病理学观察目标细胞的生长状态，免疫组织化学法检测Ⅷ因子、CD31、CD34在细胞中的表达以鉴定目标细胞。结果应用胰蛋白酶、胶原酶二步消化法可成功获取人视网膜血管内皮细胞，原代培养的细胞72h贴壁，第9～10天细胞达到融合状态，呈铺路石样。常规组织学观察显示，细胞核呈鲜亮蓝色，细胞质呈淡红色。培养的细胞对Ⅷ因子、CD31、CD34相关抗原表达呈阳性反应。结果显示培养的血管内皮细胞均有CD31、CD34同时表达，但其阳性染色程度低于Ⅷ因子相关抗原阳性反应，人脐静脉血管内皮细胞（UVECs）与人血管内皮细胞相同。结论在胰蛋白酶、胶原蛋白酶消化法的基础上用含10％优质胎牛血清的人内皮细胞培养基中添加生长因子和肝素钠，并用FN包被培养皿进行体外培?

英文摘要：

Background To optimize the culture method of human retinal mierovascular endothelial cells is very important for the study of retinal angiogenesis disease. Human retinal microvascular endothelial cells have been successfully cultured in previous studies,hut further improvement of the culture method to harvest higher yields and purity cells is still needed. Objective This study was to design a modified method to isolate and purify human retinal microvascular endothelial cells much easily and quickly,and to compare the expression of specific markers of vascular endothelial ceils,factor VIII and CD31/CD34 in the cells. Methods The use of human donor eyeballs was approved by the Ethic Commission of Zhongshan Ophthalmic Center of Sun Yat-sen University. The retina tissue from healthy donor was isolated and digested by the two-step digestion method with 2% trypsin and 0. 133% collagenase IV. Human retinal microvascular endothelial cells were collected and plated in 60 mm dishes coated by 0. 1% fibronectin and cultured in endothelial cell-specialized medium supplemented with 10% fetal bovine serum,0. 3 mg/L β-endothelial cell growth factor （ECGF） and 100 ng/L sodium heparin. During the culturing, the growth situation of the cells was monitored by morphological observation,and immunohistochemical staining was performed to probe vascular endothelial cell-specific membrane protein CD31, CD34 and factor VIII for identification of the cell purity. Results Human retinal microvascular endothelial cells were isolated successfully from the retina by the two- step digestion method. The primary cultured ceils adhered to well 72 hours later and achieved confluence with the typical cobblestone appearance 9 to 10 days after cultured. The cells exhibited the blue nuclei and reddish cytoplasm by regular haematoxylin and eosin stain and showed a strong positive response for CD31 , CD34 and factor VIII by immunohistochemistry. The positive dye of CD31 and CD34 was lower than Ⅷ factor in both endothelial cells. Conclusions Modified