大鼠视网膜微血管周细胞的选择性培养  

Selective culture of rat retinal microvascular pericytes

作　　者：刘光辉[1] 孟春[2] 徐朝阳[1] 刘安[1] 黄以鹏[2] 黄陈稳 

机构地区：[1]福建中医药大学附属人民医院眼科,福州350004 [2]福州大学生物科学与工程学院生物工程系,350108

出　　处：《中华实验眼科杂志》2014年第1期18-22,共5页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目（81102619）;福建省自然科学基金项目（2011J01197）

摘　　要：背景视网膜微血管周细胞（RMPs）在糖尿病视网膜病变（DR）等视网膜新生血管性疾病中的作用日益受到重视，并被视为潜在的治疗靶点。然而因组织材料来源受限及细胞分离纯化不易，RMPs体外研究工作的开展受到了限制。目的建立简便的大鼠RMPs分离、培养和纯化方法。方法摘取清洁级健康雄性SD大鼠的眼球，置于体积分数75％乙醇中浸泡1min，用眼科显微手术器械分离获取视网膜并将其剪成碎片，依次用2．5g／L胰蛋白酶和2g／LⅠ型胶原酶各消化15min，消化后分别过直径100μm和55μm滤网，收集视网膜消化后的微血管片段，用含有体积分数20％胎牛血清的低糖型DMEM接种于6孔板，至培养14～16d细胞达到80％～90％融合时进行消化传代，培养和传代期间通过换液法及差异消化法去除杂质细胞。用倒置相差显微镜观察细胞的形态和生长状态，用免疫荧光法检测细胞中α-平滑肌肌动蛋白（α—SMA）、血小板源性生长因子受体-β（PDGFR—β）、von Willebrand因子（vWF）、胶质纤维酸性蛋白（GFAP）的表达，以鉴定RMPs。结果RMPs在接种后24—48h自微血管片段中迁移出，7d后形成中等大的细胞集落，14～16d后达到80％-90％融合状态。RMPs传代后生长速度加快，至12—14d达到融合。形态学鉴定显示，培养的细胞呈宽大、扁平、不规则形并伴多个突起的典型周细胞形态特征，且传代至第9代形态特征无明显变化。免疫荧光法检测显示，所培养的细胞均不表达内皮细胞特异性标志物vWF，约96％的细胞对周细胞标志物α—SMA或PDGFR—β抗体呈阳性反应，极少量细胞对胶质细胞特异性标志物GFAP抗体呈阳性表达。结论本实验成功建立了一种简单、经济的大鼠RMPs分离、纯化、培养方法，可以获得理想的高纯度RMPs。Background Retinal microvascular pericytes （RMPs） have been played increasing attention as an emerging key in pathogenesis of various retinal angiogenie diseases including diabetic retinopathy, and RMPs are thought to be a potential target for treatment. Yet the study has been hindered by the difficulty of obtaining source of tissue and isolating pure population. Objective This study was to establish a simple method of isolation, purification and cultivation of primary RMPs for rat. Methods Eyeballs were extracted from clean male Sprague Dawley rats and immersed by 75% alcohol for 1 minute. The retinas were isolated and mechanical moreel. Trypsin （2.5 g/L） was firstly used and followed by type I collagenase （2 g/L） for the digestion of the retina for 15 minutes, respectively. Retinal mierovaseular fragments were screened by 100 μm and 55 μm filter screen. DMEM containing 20% fetal bovine serum was added for the cultivation and passaged of the cells. The cells were purified by exchanging medium and partial enzymatic digestion. The morphology and growth status were monitored under the phase contrast microscope,and α-smooth muscle actin （α-SMA）, platelet-derived growth factor receptor-β （PDGFR-β）, von Willebrand factor （vWF） ,glial fibrillary acidic protein （GFAP） antibodies were used for the identification of RMPs. Results RMPs migrated out of fragments after 24-48 hours of plating. On day 7, RMPs appeared in primary cultures as loose colonies. The cells reached confluence to about 80% -90% on day 14-16. The subcultures grew faster than the primary and reached confluence on day 12-14. The culture showed typical morphology of pericyte with large irregular triangular cell body and multiple long processes, and they could be repeatedly passaged 9 times without obvious loss of characteristic phenotype. Fluorescence assay exhibited that 96% of the cells showed positive immunofluorescence for α-SMA and PDGFR-β,confirming the purity of RMPs in culture. However,only a few of them were pos

关 键 词：视网膜 微血管 周细胞 分离 纯化 培养 鉴定 

分 类 号：R774.1[医药卫生—眼科]