缺氧条件下共培养时观察Müller细胞对RPE细胞生长的影响

Effects of Miiller cells on growth of RPE cells in a co-culture system in hypoxia condition

背景视网膜色素上皮（RPE）细胞和Mtiller细胞在维持视网膜的正常代谢中起重要作用，多种生理、病理过程与二者的相互作用有关。目的利用Transwell小室体外共培养兔Mtiller细胞与RPE细胞，观察缺氧条件下Moller细胞对RPE细胞增生和迁移的影响。方法应用组织块培养法和酶消化法分别培养原代兔Mtiller细胞和RPE细胞，免疫组织化学法鉴定2种细胞。取第3代培养良好的细胞进行试验。应用CoCl2培养细胞以建立细胞化学缺氧模型，分为常氧组和缺氧组，每组又分为对照组（RPE细胞单独培养）、共培养组（MOiler细胞与RPE细胞共同培养）。利用Transwell小室建立Mtiller细胞和RPE细胞的共培养模型，各组分别培养3、6、24、48h，利用MTT法和结晶紫染色法对培养存活的RPE细胞进行计数，检测常氧及缺氧条件下Miiller细胞对RPE细胞增生和迁移的影响。结果原代培养的RPE细胞符合RPE细胞的特征，90％以上呈现CK．18阳性反应。培养的Miiller细胞对胶质纤维酸性蛋白（GFAP）和S-100染色呈阳性反应。与常氧培养条件比较，缺氧条件下单独培养的RPE细胞增生数量增加，细胞迁移数量增加，差异有统计学意义（P〈0．01）；缺氧条件下与MUller细胞共培养的RPE细胞增生数量和迁移数量明显增加，与常氧组和单细胞培养情况下比较，差异均有统计学意义（P〈0．01）。缺氧条件下，与RPE细胞单细胞培养组比较，共培养组RPE细胞的增生和迁移增加，差异均有统计学意义（P〈0．01）。结论缺氧可以促进RPE细胞的增生和迁移，Mtiller细胞可以加重缺氧引起的RPE细胞的增生和迁移。

Background More and more evidences suggest that the interaction of retinal pigment epithelial （RPE） cells and Mtiller cells plays an important role in the progression of retinal diseases. Objective Present study was to investigate the role of Mtiller cells in growth of RPE cells with a co-culture system in vitro under the hypoxia condition. Methods Mtiller cells and RPE cells of rabbit were primarily cultured and digested using explant culture method and trypsas method. Cultured Mtiller cells and RPE cells were identified by glial fibrillary acidic protein（GFAP） staining,S-l00 staining and cytokine-18 staining respectively. CoC12 was added into DMEM to establish the hypoxia models of Muiller cells and RPE cells. The third generations of Mtiller cells and RPE cells were co-cultured under the normoxia and hypoxia using transwell chamber, and the proliferation and migration of cultured RPE cells were examined using MTT and compared with only RPE ceils cultured group at 3, 6, 24 and 48 hours. Results Over 90% of primarily cultured RPE cells showed the positive response for CK-18. Cultured Mtiller cells presented the positive response for GFAP and S-100. The proliferation and migration of RPE cells were significantly increased in hypoxia condition compared with normoxia condition （P〈0.01）. In hypoxia group, amount of proliferation and migration of RPE cells in co-culture group were higher than the only RPE cells cultured group （P〈0. 01 ）. Conclusion Hypoxia condition promote the proliferation and migration of RPE cells. Mailer cells appear to aggravate the proliferation and migration of RPE cells under the hypoxia status.