猪视网膜色素上皮细胞的体外培养与鉴定

目的建立猪视网膜色素上皮（RPE）细胞体外分离、纯化培养体系并进行鉴定,为研究RPE细胞的功能及治疗相关眼底疾病提供细胞来源。方法采用胰蛋白酶消化法获取猪RPE细胞进行原代及传代培养,倒置显微镜观察细胞生长过程及形态变化并计算生长数据,角蛋白和RPE65蛋白抗体免疫细胞化学染色鉴定细胞。结果用胰蛋白酶消化法分离培养的猪RPE细胞生长良好,表现为多边形和梭形两种形态,原代细胞胞浆内含有丰富的色素颗粒,传代细胞内色素颗粒较原代细胞少。免疫组织化学法证实RPE细胞均为鼠抗人角蛋白染色阳性。结论培养的细胞可作为体外猪RPE细胞的来源,体外分离、纯化和培养猪RPE细胞的成功,为RPE细胞功能和相关视网膜疾病的深入研究提供了细胞来源。

Culture of pig retinal pigment epithelial cell in vitro

Objective The function and structure of retinal pigment epithelial（RPE） cells have been well-known.To establish the cultivating approach of animal RPE cells is very important for the experimental and clinial research on retinal disease.This study was to create a cultivating method of pig RPE cells.MethodsRPE cells were isolated from 20 pig eyes and digested with trypsin.The cells were cultured in DMEM-F12 medium containing 10% fetal bovine serum and passaged in a 2-or 3-day interval.The growth curve of the RPE cells was drawn for the 1st,3rd and 7th generation of cells,and the morphological features and differentiation of RPE cells were observed under the phase contrast microscope.The cultured RPE cells were identified by immunohistochemical staining with anti-human keratin and RPE65.ResultsThe primary RPE cells were round in shape and presented with affluent intracellular melanin granules,indicating a good growth in vitro.Good growth curves were seen in the 1st and 3rd generation of cells.Transparent shape and reduced melanin granules of cultured RPE cells were found with the prolong of differentiation time.Immunohistochmical staining demonstrated that the RPE cells were presented the brown staining for anti-human keratin and RPE65 in the cytoplasm.ConclusionThe cultured cells can be used as the source of pig RPE cells.The separation and purification of the pig RPE cells in vitro provide a possible way for the research on RPE cells and related retinal diseases.