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背景 在研究视网膜Müller细胞的病理生理过程中,建立Müller细胞的培养模型、获得高纯度的Müller细胞是基本步骤.目前使用的纯化培养Muller细胞的技术所获得的细胞纯度不够理想.目的 建立一种获得高纯度原代培养视网膜Müller细胞的技术方法.方法取5只新生SD大鼠的视网膜经质量分数0.01%胰蛋白酶消化制备细胞悬液,然后在含质量分数10%胎牛血清的DMEM培养基中进行原代培养,细胞扩增至大于6×105个时收集细胞,无菌条件下用流式细胞仪进行分选,将细胞悬液中体积最大、数量最多的一种细胞分选出来继续培养并传代,应用透射电镜和光学显微镜观察细胞的形态学变化,应用免疫组织化学技术鉴定培养和传代的细胞类型及纯度.结果 原代培养视网膜Müller细胞成功,其中混杂有其他类型的小细胞,生长缓慢,培养3周生长速度加快.用分次贴壁法清除成纤维细胞,经传代去除部分神经元,经流式细胞仪纯化后可获得细胞成分单一、体积最大的一批细胞.透射电镜下细胞内可见丰富的线粒体、高尔基体和大量直径8～10 nm的微丝.培养细胞的胶质纤维酸性蛋白(GFAP)免疫组织化学染色细胞阳性率为100%.结论 利用流式细胞仪对培养的Müller细胞进行分选是可行的,能够获得高纯度的视网膜Müller细胞.“,”Background Establising the culture model of Müller cells for obtaining the highly putified target cells is essential for the study about the physiology and pathology of retinal Müller cells. The exsiting purifing method for culturing Müller cells is dissatisfactory. Objective This study was to establish a method to obtain high purifing Müller cells. Methods The retina from 5 clean newborn SD rats were isolated and digested by 0. 01% trypsin and cultured in DMEM containing 10% fetal bovine serum. The cellular suspension was then prepared,and the target cells were screened using flow cytometry based on the size and the quantity of cells. Cultured and passaged cells were identified by transmission electron microscope and light microscope. Immunocytochemistry was used to detecte the expression of GFAP in cultured cells for the determination of type and purity of the cells. Results The cells showed the similar shape to retinal Müller cells after primarily culture with the large volume, and some small other types of cells could been seen. The growth of cells was quickly 3 weeks later. The fibroblasts were removed using sticking-wall by steps,and neurons were eliminated following passage. Aboundent of cellular organs were seen under the transmission electron microscope. The positive response rate of the cells for CFAP was 100%. Conclution Flow cytometry offer a rapid and feasible approach for purifying Muller cell and it builds the foundation for further study about Müller cells.