体外培养人视网膜微血管内皮细胞屏障功能的研究  

Barrier function of human retinal microvascular endothelial cells cultured in vitro

作　　者：李建桥[1,2] 江静波[3] 罗燕[1] 刘清云[1] 肖伟[1] 史煜[1] 马红婕[1] 唐仕波[1] 

机构地区：[1]中山大学中山眼科中心,国家眼科学重点实验室,广东广州510060 [2]山东大学齐鲁医院眼科,山东济南252000 [3]广东省妇幼保健院新生儿科,广东广州510010

出　　处：《中国病理生理杂志》2009年第4期749-754,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30772392No.30872819);卫生部临床学科重点资助项目(No.2007353)

摘　　要：目的:研究原代培养的人视网膜微血管内皮细胞屏障功能。方法:对人视网膜微血管内皮细胞进行原代培养并鉴定,第4代细胞接种于Polyester(PET)膜上,不同时点对滤膜表面行显微镜观察,通过紧密连接相关蛋白occludin的表达,间接反映细胞之间紧密连接的形成,应用共聚焦免疫显微镜观察2、3、4周occludin表达的变化;跨膜电阻(TER)检测屏障的稳定性,并通过测量正常培养条件和5μg/L的血管内皮生长因子(VEGF)干预下辣根过氧化物酶(HRP)通透性的改变反映屏障特性。结果:成功培养了人视网膜微血管内皮细胞,纯度可达95.8%。细胞接种1周后融合为单层,接种后2、3、4周细胞密度无明显变化,细胞接触紧密,呈单层生长,可见occludin在细胞相邻边界规则的表达;occludin在2、3、4周,在细胞内的分布逐渐移向相邻细胞交界处;2周时视网膜微血管内皮细胞跨上皮细胞电阻达(120.62±3.97)Ω/cm2,2、3、4周差异无显著(P>0.05);单层细胞的通透性检测结果显示,HRP从滤膜上方到达下方随时间延长呈线性增加,且在VEGF的干预下,各时点通透率明显增加(P<0.05)。结论:利用体外培养的人原代视网膜微血管内皮细胞建立单层细胞具有典型的屏障功能特性,可作为体外研究血视网膜内屏障的有用模型。AIM： To investigate the blood -retinal barrier （BRB） function of human retinal microvascular cells （hRMECs） cultured in vitro. METHODS： Primary hRMECs were cultured and identified. The fourth generation was inoculated in a micro porous filter with the filter membrane of Polyester （PET）. Cells on the surface of filter membrane was observed by phase microscope at different time points, and occludin staining was used to show the tight junction （TJ） indi- rectly. Transendothelial electrical resistance （TER） was detected at 2, 3,4 and 5 weeks, respectively and the permeability was measured with horseradish peroxidase （HRP） in both normal condition and stimulated with 5 μg/L VEGF 15 min, 30 min, 45 min, 60 min at 2 weeks monolayer. RESULTS： Primary hRMECs were cultured successfully and purity was up to 95. 8%. Cells formed a monolayer 1 week after seeding. 2, 3 and 4 weeks after inoculation, the density of hRMECs stayed unchanged. Occludin staining showed TJ indirectly 2 weeks after inoculation. Distribution of occludin showed the TJ maturing with time delaying. TER reached （ 120. 62 ± 3. 97 ） Ω/cm2 at 2 weeks and kept stable within 4 weeks （ P 〉 0. 05 ）. VEGF at the concentration of 5 μg/L increased HRP penetration significantly compared to that in normal culture condition （P 〈 0. 05 ）. CONCLUSION： The filter of PET membrane may be a useful method to set up the model monolayer of hRMECs and investigate the barrier function of blood retina barrier.

关 键 词：血管内皮细胞 细胞培养 血视网膜屏障 

分 类 号：R363[医药卫生—病理学]