摘要
目的 体外培养人视网膜微血管内皮细胞,探讨重组rAAV2-色素上皮衍生因子（PEDF）对该细胞在不同氧环境下增殖的影响.方法 实验研究.2%胰蛋白酶和0.1%Ⅱ型胶原酶消化视网膜,获得视网膜微血管内皮细胞;接种于预先包被纤维连接蛋白的培养瓶内,用含10%胎牛血清、内皮细胞生长因子、胰岛素-转铁蛋白-硒添加物的内皮细胞培养基,置于5%CO237 ℃培养箱内培养.相差显微镜观察细胞形态及生长情况.抗第Ⅷ因子相关抗原抗体鉴定内皮细胞.以125 μmol/L CoCl2建立HRCECs化学低氧模型,观察低氧对内皮细胞增殖的影响.按照105病毒基因组数/细胞的剂量进行rAAV2-PEDF病毒转染,分别设空白和阴性对照,激光共焦显微镜下观察EGFP阳性细胞,免疫印迹法检测PEDF蛋白表达.MTT法测定rAAV2-PEDF对不同氧条件下对细胞增殖的影响.流式细胞仪检测rAAV2-PEDF对不同氧条件下对细胞凋亡的影响.两组间比较采用独立样本t检验,多组间比较采用方差分析.结果 原代培养的人视网膜微血管内皮细胞48～72 h贴壁,2周左右细胞融合.第Ⅷ因子相关抗原鉴定细胞呈阳性.转染病毒48 h后,激光共焦显微镜下观察可见EGFP阳性细胞,免疫印迹法检测,实验组PEDF表达明显强于其他组.MTT结果显示,单纯低氧处理,正常对照组A值为0.085±0.021,实验组A值为0.166±0.024（t=3.938,P＜0.05）.rAAV2-PEDF干预正常氧条件下人视网膜微血管内皮细胞,A值分别为：正常对照组0.171±0.011,rAAV2-EGFP组0.178±0.016,rAAV2-PEDF组0.169±0.017（F=0.01,P＞0.05）.rAAV2-PEDF干预低氧条件下人视网膜微血管内皮细胞,A值分别为：单纯CoCl2组0.166±0.013,CoCl2＋rAAV2-EGFP组0.155±0.012,CoCl2＋rAAV2-PEDF组0.116±0.015（F=6.25,3.50;P＜0.05）.rAAV2-PEDF干预正常氧条件下人视网膜微血管内皮细胞,正常对照组凋亡细胞比例为2.3%,rAAV2-EGFP组为3.3%,rAAV2-PEDF组为1.7%.rAAV2-PEDF干预低氧条件下人视网膜�
Objective To culture human retinal capillary endothelium cells（HRCECs） in vitro and explore the effect of rAAV2-PEDF on proliferation of HRCEs. Methods Retinas were digested by 2. 5%trypsin and 0.1% collagenase Ⅰ in order. The isolated cells were cultured on fibronectin-coated dishes in media of human endothelial-sFM basal growth medium （HE-SFM BGM） with 10% fetal bovine serum,insulin-transferin-selenium （ITS） and endothelial cell growth factor （ECGF）. The cultured cells were identified by anti-factor Ⅷ related antigen though immunohistochemistry stain. The effect of hypoxia induced by CoCl2 on proliferation of HRCECs was assessed by MTT assay. After rAAV2-PEDF were transfected into HRCECs, the EGPF positive cells were observed by laser confocal scanning microscop, the protein expression of PEDF were detected by Western blot, and the proliferation of HRCECs were checked by MTT assay. Flow cytometry was used to analyze the apoptosis of HRCECs. Results Cultured HRCECs attached in the bottom of dishes in 48 h-72 h and grew to confluence in 2 weeks after seeding. HRCECs were with a positive brown staining for factor Ⅷ. EGPF positive cells were seen under laser confocal scanning microscop after 48h of rAAV2-EGFP transfection. The expression level of PEDF protein was higher in experimental group than in control group. The results of MTT assay showed the numeric value OA was 0.085±0.021 in normal group and 0.166±0.024 in hypoxia group（t =3.938,P〈0.05）. In normal oxygen condition, the numeric value OA was 0.171±0.011 in normal control group, 0.178±0.016 in rAAV2-EGFP treated group, and O. 169±0.017 in rAAV2-PEDF treated group（F=0.01 ,P〉0.05）. In hypoxia condition, the numeric value OA was 0.166±0.013 in CoCl2 treated group, 0.155±0.012 in CoCl2 ＋ rAAV2-EGFP treated group, and 0.116±0.015 in CoCl2 ＋ rAAV2-PEDF treated group. In normal oxygen condition, the ratio of apoptosis was 2. 3% in normal contral group,and 3. 3% in rAAV2-EGFP treated group,and 1.7% in rAAV2-PEDF treated
出处
《中华眼科杂志》
CAS
CSCD
北大核心
2011年第3期197-201,共5页
Chinese Journal of Ophthalmology
基金
基金项目：国家自然科学基金（30901642）
广东省自然科学基金（8151008901000174）
