兔眼与猪眼视网膜色素上皮细胞分离及培养方法的对比

目的分别对兔眼及猪眼视网膜色素上皮（RPE）细胞进行体外分离、培养和鉴定，探讨二者的异同点及注意事项。方法对有色兔眼和猪眼的RPE细胞采用胰蛋白酶消化法进行分离、培养和传代，取第4代细胞用于实验。免疫细胞化学染色对传代的细胞进行鉴定。光学显微镜下观察并比较培养的2种动物来源RPE细胞的形态、特征，并对2种动物来源的RPE细胞的培养过程进行比较。结果2种动物来源的RPE细胞分离方法的不同与各自眼球的解剖结构相关，主要体现在消化过程，猪眼RPE细胞1次消化即可分离，兔眼RPE细胞需2次消化得到。原代猪眼RPE细胞24h内贴壁较兔眼48～72h贴壁时间早。培养早期细胞生长活跃，随传代次数增加活力下降，色素颗粒减少。免疫细胞化学染色提示细胞角蛋白（AE1／AE3）表达阳性。结论2种动物来源的RPE细胞均可通过胰蛋白酶消化法分离，获得的RPE细胞数量多，培养成功率高。猪眼RPE细胞的分离培养更为容易。4代以内的RPE细胞适合于实验研究。

Comparison of in vitro isolation and culture of retinal pigment epithelial cells in rabbit eye and pig eye

Background　Culture of retinal pigment epithelium(RPE) cells is very important for establishment of proliferative vitreoretinopathy (PVR) model,prevention and treatment of PVR as well as RPE cell transplantation.Isolation of animal RPE cells by trypsinization is a critical step.ObjectiveThe present study is to establish the methods of isolation and culture of retinal pigment epithelium (RPE) cells in rabbit and comparied with that of pig RPE culture.MethodsRPE cells were isolated by trypsinization in pigmented rabbit and pig and cultured in DMEM containing 20% fetal bovine serum.Cultured RPE cells were identified by immunochemistry.The fourth generations of cells were used in this experiment.Morphology and characteristics of cultured RPE cells from rabbit and pig were examined and compared under the light microscope.ResultsIsolated RPE cells from pig were obtained by once trypsin digestion,but two times of trypsinization were needed in rabbit RPE cells isolation.The differentiation in response to trypsinization was related to anatomic difference between the two types of cells .The adherence time of pig RPE cells was 24 hours ,however,the rabbit RPE needed 48-72 hours after culture.Proliferation and vitality of cultured cells were gradually attenuated and melanin decreased after several times subculture.The morphology of culture RPE cells was obviously different between rabbit and pig because species difference.Immunohistochemistry demonstrated the positive response of RPE cells for keratin.ConclusionRPE cells can be acquired from both rabbit and pig by trypsinization and culture.The culture process of RPE cells of pig is simpler than that of rabbit.Cells within the fourth generations are suitable for experimental application.