酶-机械分离-酶消化法分离培养兔眼虹膜色素上皮细胞

Isolation and culture of rabbit's iris pigment epithelial cells by enzyme-microdissection-enzyme digestion method in vitro

目的 建立兔眼虹膜色素上皮细胞体外培养体系。方法 采用酶 机械分离 酶消化法分离兔眼IPE细胞 ,台盼蓝染色法测定细胞活力 ,在体外对其进行原代及传代培养扩增 ,倒置相差显微镜观察IPE细胞的生长情况。取第 2次传代的IPE细胞行AE1/AE3及S 10 0抗体细胞免疫组化染色对其进行鉴定。结果 酶 机械分离 酶消化法分离得到IPE细胞的活力为 70 %～ 85 % .原代细胞 16～ 4 8h贴壁 ,72h后开始生长 ,细胞大多呈多角形或方形 ,少数为梭形 ,12～ 16d生长融合为单层细胞。第 1次传代的IPE细胞 6～ 12h贴壁 ,5～ 7d生长融合为细胞单层。IPE细胞在体外培养可传 5～ 6代。免疫组化染色显示 ,几乎所有细胞胞浆呈现棕黄色阳性反应 ,对照组胞浆不染色。结论 酶 机械分离 酶消化法分离培养兔眼IPE细胞易于操作 ,联合应用细胞角蛋白、S 10 0蛋白单克隆抗体的免疫组化技术可用来鉴定IPE细胞。

Objective To establish iris pigment epithelial (IPE) cells culture system of rabbits in vitro. Methods IPE cells were isolated by the enzyme- microdissection-enzyme method and IPE's viability was tested by trypan blue. We cultured, subcultured and proliferated IPE in vitro.Cultured cells were ob served by phase contrast microscopy and IPE cells of passage two were identified by immunocytochemistry. Results IPE cells's viability was 70%～85% isolated by enz yme-microdissection-enzyme method.The primary cultured cells attached in 16～4 8 hours and spread after 72 hours.Most of them were polygonal or cubical and be came confluent 12～16 days after plating.IPE cells of passage one attached in 6 ～12 hours and became confluent monolayer cells in 5～7 days. IPE cells could be cultured for 5 to 6 generations in vitro.AE1/AE3, S-100 immuncytochemical anal ysis of IPE (p2) showed that almost all of the IPE cells were stained yellow-br own, while IPE cells of control group were not stained. Conclusion Through combining AE1/AE3 with S-100, IPE cells could be identified by immunocy tochemistry.