体外人羊膜培养兔角膜缘上皮细胞的细胞形态与连接复合体形成的研究

Morphologic characteristics and formation of adhesion complex of cultured rabbit limbal epithelium on amniotic membrane in vitro

目的观察体外人羊膜上培养出的兔角膜缘上皮细胞形态特征及与羊膜基底膜连接复合体的形成。方法将15只新西兰白兔角膜缘组织（2mm×2mm），于37℃ 5％ CO2孵箱中用1．2U／ml裂解酶Ⅱ处理20min，之后在人羊膜上皮面先行浸液培养2周，后行气-液培养2周。于培养期间，每周定期进行透射电镜检查，以观察连接复合体的形成过程，培养至第4周后，采用AE5免疫组织化学、高碘酸．希夫、苏木精-伊红等染色方法鉴定分化的上皮细胞，鉴别杯状细胞，观察细胞的形态特征。结果兔角膜缘上皮细胞体外培养10—14d可充满人羊膜面，对AE5免疫组织化学方法染色分化出的上皮细胞均呈阳性而高碘酸．希夫染色均呈阴性。苏木精-伊红染色显示从兔角膜缘上皮细胞分化出的上皮细胞与正常兔角膜上皮细胞相比无明显差异；自培养至第14天均未见与羊膜基底膜的连接复合体形成；于第21天时仅观察到初始阶段的连接复合体，第28天时仍未见明显变化。结论于体外人羊膜上培养的兔角膜缘上皮细胞分化出的细胞是角膜上皮细胞，其形态与正常兔角膜上皮细胞相同，并与羊膜基底膜仅形成初始阶段的连接复合体。

Objective To investigate the morphologic characteristics and the formation of adhesion complex formation of cultured rabbit limbal epithelium on amniotic membrane in vitro. Methods The limbal explants（size：2 mm ×2 mm） were taken from 15 rabbits and were treated with 1.2 U/ml dispase Ⅱ at 37℃, in 5% CO2 incubator for 20 min, then, cultured on amniotic membrane in vitro for 4 weeks. It was cultured in culture medium during the first two weeks, and then cultured as airlifted culture in the following two weeks. Transmission electronmicroscope （TEM） was performed weekly, the AE-5 immunohistochemical staining, PAS staining and HE staining were performed at the end of fourth week. Results It took 2 weeks for the cultured limbal epithelium to cover the whole amniotic membrane. Immunocytochemical studies showed that cultured limbal epithelium was positive for keratin 3 and negative for PAS staining. HE staining showed that the morphologic characteristics of cultured limbal epithelium were similar to normal rabbit corneal epithelium. The adhesion complex formation didn＇t appear on 7th day and 14th day. Early stage of adhesion complex formation appeared on 21st day, and without significant change until 28th day. Conclusions The morphologic characteristics of cultured limbal epithelium on amniotic membrane in vitro are similar to normal rabbit corneal epithelium. Only early stage of adhesion complex is formed as compare to the normal adhesion complex of rabbit.