新生大鼠视网膜Müller细胞原代培养方法的改良

Modified procedure for primary culture of retinal Miiiler cell in newborn rat

背景目前，视网膜Muller细胞的干细胞特性日益受到关注，优化Muller细胞的培养方法对视网膜干细胞治疗的进一步研究具有重要意义。目的对视网膜Muller细胞的原代培养方法加以改良，建立一种简单、有效的实验室分离纯化Muller细胞的方法。方法取新生SPF级SD大鼠眼球，培养基浸泡后室温条件下避光过夜，显微镜下分离视网膜，直接吹打成微小组织悬液后接种于培养瓶中，8～10d后行第1次换液，之后2～3d换液1次，至细胞完全融合后进行传代。细胞形态一致后于光学显微镜下观察细胞的形态特征，免疫荧光法检测Muller细胞标志物谷氨酰胺合成酶（GS）和波形蛋白（Vimentin）的表达，进行细胞鉴定；采用流式细胞术对所得细胞进行纯度检测。结果原代培养的Muller细胞胞体狭长，细胞质丰富。传至3代的细胞95％以上对GS和Vimentin反应阳性，阳性染色主要位于细胞质和细胞核。Vimentin阳性染色主要位于细胞质。流式细胞仪检测显示传3代后99．7％的细胞GS表达阳性。结论采用Muller细胞改良法一流式细胞术对所得细胞进行纯度检测，简单可行，收获的细胞数量多、纯度高。

Background Mtiller cells has been recognized as being vital in both healthy and diseased retina. Recently, these cells even have been identified to be the source of retinal progenitor cells. In order to study the possible function of the retinal Muller ceils, it is important to establish a practical procedure to obtain the purified cells. Objective This study was to simplify the procedure of primary culture and purification of retinal Mtiller cells in vitro. Methods Eyeballs of SPF newborn SD rats were enucleated and retinas were dissected free after soaking the eyeballs overnight in DMEM/F12 medium in room temperature. Then the retinas were mechanically dissociated into micro aggregates and cultured in DMEM/F12 medium containing 10% FBS for 8-10 days. The floating retinal aggregates and debris were removed and the medium was changed in 2-3 days interval to get more purified flat cell population. Cultured cells were passaged after confluent. [mmunofluorescence staining was used to detect the response to glutamine synthetase （GS） and Vimentin for the identification of the cultured MUller cells, and flow cytometry （FCM） was used to estimate the purity of the cells. Results Cultured Muller cells had large cellular body and richer cytoplasm. More than 95% of the cells showed the positive response for GS with the brown staining in cytoplasm and cellular nuclei,and the positive staining also was seen for Vimentin in cytoplasm. FCM showed that 99.7% of the cells were GS positive after 3 passages. Conclusions Modified procedure in this experiment is a simple and practical method for culturing retinal Muller cells.