气液界面培养体外构建角膜上皮的实验研究

Air liquid interface cultivation of corneal epithelium constructed in vitro

目的:建立气液界面培养体外构建兔角膜上皮的方法。评价体外构建角膜上皮的生物学特性。方法:新西兰白兔15只30只眼,取角膜缘上皮组织。组织块接种原代培养。细胞融合生长后,消化传代。以去上皮细胞人羊膜组织为载体,接种培养传代细胞,细胞融合后,用气液界面培养方法继续培养周。倒置显2微镜对体外构建的角膜上皮进行观察。培养细胞膜片固定,苏木精-伊红(H)E染色;AE1细胞角蛋白单克隆抗体免疫组化染色,光镜观察。常规制作电镜标本,透射电镜观察。以传统培养方法作为对照。结果:倒置显微镜观察,气液界面培养可以形成连续的上皮细胞层,细胞和载体之间连接较牢固。HE染色显示体外构建的角膜上皮具有复层扁平上皮的结构。免疫组化染色有较高的阳性率。透射电镜观察细胞之间有丰富的桥粒样连接。结论:气液界面培养方法可以构建复层鳞状角膜上皮。与传统培养方法比较,构建的角膜上皮组织具有较优越的生物学特性。

AIM:To establish a method of air liquid interface culture for construction of the rabbit corneal epithelium in vitro, and evaluate its biological characteristics. METHODS:Epithelial tissue of corneal limbus was harvested from 30 eyes of 15 New Zealand rabbits. Explants were incubated for primary culture. The cells had digestive transfer culture after fusion growth. Then the cells were cultivated on a carrier of modified human amniotic membrane,and cultured by using air liquid interface culture for 2 weeks after fusion.The corneal epithelium cells,constructed in vitro,were observed under inverted phase contrast microscope.The cultured limbal epithelial cell sheets were fixed and stained with hematoxylin eosin(HE) sing;AE1 cytokeratin monoclonal antibody was performed immunohistochemical staining and examined under light microscope.Routine electron microscope samples were made and observed under the transmission electron microscope (TEM).The air liquid interface culture technique was compared with the conventional culture method. RESULTS:The results of the inverted phase contrast microscope showed that successive layers of epithelial cells could be cultured on the air liquid interface,and the joint of cell and carrier was firm. HE staining showed that there was stucture of stratified squamous epithelium in the corneal epithelium which was constructed in vitro. Immunohistochemical staining had higher positive rate.More desmosomal junctions between the cells were observed under the TEM. CONCLUSION:A multi layered rabbit corneal epithelium is successfully construction in vitro by a air liquid interface culture technique, and the constructed corneal epithelium had superior biochemical characteristics to those cultured with the conventional method.