Müller细胞对大鼠视网膜微血管内皮细胞增生和迁移的影响  

Effects of Müller cells on proliferation and migration of rat retinal microvasc- ular endothelial cells

作　　者：郭斌[1] 王应利[1] 惠延年[1] 王雨生[1] 马吉献[1] 

机构地区：[1]第四军医大学西京医院眼科,中国陕西省西安市710032

出　　处：《国际眼科杂志》2006年第2期345-351,共7页International Eye Science

摘　　要：目的:用免疫磁珠法原代培养大鼠视网膜微血管内皮细胞(RMEC),观察共培养Müller细胞对视网膜微血管内皮细胞增生和迁移的影响。方法:采用免疫磁珠的细胞分选法分离大鼠视网膜微血管内皮细胞,在条件培养基中培养生长,采用第Ⅷ因子抗体免疫组化染色方法鉴定细胞,采用流式细胞术和台盼兰染色分析传代中的微血管内皮细胞纯度以及细胞活性。传统的方法培养并鉴定Müller细胞。采用不同孔径微孔滤膜培养小室,进行大鼠视网膜微血管内皮细胞与Müller细胞分离共培养,对照组培养环境中无Müller细胞。以细胞曲线描绘反映细胞增生,并用流式细胞仪测定细胞周期。相差显微镜下计算穿过微孔滤膜迁移贴附于背面的内皮细胞数量。结果:通过磁珠分离方法获得了纯度为97.13%大鼠视网膜微血管内皮细胞,细胞活力达92%,第Ⅷ因子抗体染色呈阳性。与Müller细胞共培养的RMEC可早于正常培养2～3d进入生长平台期。RMEC中S期和G2期比例增加,G1期比例相对减少。S、G2和G1期百分比,在共培养组24h分别为44.0%,11.2%和44.8%,48h分别为42.3%,10.9%和46.8%;对照培养组24h分别为41.3%,4.9%和53.8%,48h分别为40.1%,4.7%和55.2%。迁移的细胞数增加,共培养6h移行至膜下的内皮细胞数12.2±2.5个,12h为51.7±23.4个,对照培养6h移行至膜下的内皮细胞3.3±2.5个,12h为14.7±7.0个,6h和12h两组内皮细胞数差异具有统计学意义(P<0.05)。结论:免疫磁珠细胞分选方法可以获得高纯度的大鼠视网膜微血管内皮细胞,且对细胞活力无影响。Müller细胞可以促进视网膜内皮细胞的迁移,促进该细胞的增生。AIM： To culture primary retinal microvascular endothelial cells （RMEC） from Wistar rats with magnetic cell sorting （MACS） system and to observe the effects of cocultured Müller cells on proliferation and migration of RMEC. METHODS： Rat RMEC were isolated with MACS system and cultured in conditioned medium with endothelial cell growth supplements. These cells were characterized for expression and localization of factor Ⅷ （yon Willebrand） with immunohistochemistry staining. The purity and viability of passaged RMEC were analyzed by the methods of fluorescence activated cell sorter （FACS） and trypan blue staining respectively. Müller cells were cultured and identified by conservative methods. RMEC and Muller cells were cocultured uncontactedly in special cell coculture insert systems with different micropore diameters. RNEC in control groups were cultured without Müller cells. Cell proliferations were recorded by growth curve and FACS measured cell life cycles. The number of cells which migrate through micropores and stay on the outer bottom side of insert systems were observed and calculated under invert microscopy. RESULTS： Rat RMEC could be purified from mixed cells （purity 〉97.13% ,viability 〉92% in passage six） and passaged serially. The cells of passage 2-6 were positive staining for factor Ⅷ. Compared with normal control group, Müller cells improved RMEC developing into platform stage for 3d, increased proportions of RMEC in stage S and stage G2 and decrease in stage G1. Percentage of stages S, G2 and G1 in RMEC cocultured with Müller cells were 44.0%,11.2% and 44.8% at 24h and 42.3%, 10.9% and 46.8% at 48h respectively. Percentage of stage S, stage G2 and stage G1 in RMEC in control group were 41.3%, 4.9% and 53.8% at 24h and 40.1%, 4.7% and 55.2% at 48h respectively. The amount of RMEC immigrated onto opposite side of filters in cocultured group was more than that in control group. The number of immigrated RMEC in cocultured group was 12.21 2.5 at 6h and 51.71 23.4

关 键 词：视网膜 胶质细胞 微血管内皮细胞 增生 迁移 磁珠 

分 类 号：R774.1[医药卫生—眼科] R331.32[医药卫生—临床医学]