The study of Corneal epithelial Cell Transplantation

我们尝试研制一种专为兔角膜上皮细胞设计的无血清细胞培养液。首先，当在所测试的基础培养基中研究Ca^<2+>浓度的影响时，0.03mM的Ca^<2+>浓缩物：Lon在培养的角膜上皮细胞中显示出最大的生长。因此，我们使用该Ca^2+浓度，比较了MEM、MCISBI 53、F-10、MCDB 153和F-10（1：1）、MCDB 153和MEM（1：1）之间基础培养基的生长能力。结果表明，MCDBI 53和F-10（1：1）组成的培养基中细胞生长最快。第三，使用这种补充有HEGF、氢化可的松、胰岛素、BPE（牛垂体提取物）的基础培养基，将在-70 ℃冷冻的原代培养细胞成功地传代培养至第15代。本文报道的培养液是一种上级培养兔角膜上皮细胞的基础培养基。由于在初始培养一周后成功地形成了两到三层立方上皮细胞，因此这种上皮片可以移植到体内眼表中。我们研究了在一定环境中体内结膜上皮是否真的发生生物学改变，用64 Kd角蛋白表达免疫组织化学方法检测了植入中央角膜基质袋中的结膜上皮的生物学特性，一种特异性的分化标志物角膜上皮细胞。结果表明，结膜上皮的生物学特性可以受到外部环境的影响，变得与角膜上皮相似。

We tried to develop a noble serum-free cell culture medium specially designed for rabbit corneal epithelium. First, when the effect of Ca^<2+> concentration was investigated in a basal medium tested, the Ca^<2+> concentrate : Lon of 0.03mM showed maxiTmnn growth in cultured corneal epithelial cells. Therefore, using this Ca^<2+> concentration, we compared the growth ability of basal medium among MEM, MCISBI53, F-10, MCDB153 and F-10(1 : 1), MCDB153 and MEM(1 : 1). The results indicated that the medium consisting of MCDBI53 and F-10(1 : 1)showed the highest cell growth. Third, using this basal medium supplemented with HEGF, hydrocortisone, insulin, BPE(bovine pituitary extract), primary cultured cells frozen at -70 C were successfully subcultured up to the 15th-generation. The medium reported here is presixmed to be far superior to any other available basal medium for culturing rabbit corneal epithelium.We tried to establish in vitro rabbit corneal epithelial cell layer on type IV collagen sheet. Since two to three layers of cuboidal epithelial cells were successfully made one week after initial culture, this epithelial sheet can be transplanted into in vivo ocular surface.We investigated whether biological alteration truly occurs in in vivo conjunctival epithelium in a certain environment, the biological characteristics of conjunctival epithelium implanted in a central corneal stromal pocket were immunohistochemically examined using 64Kd keratin expression, a specific differentiation marker. for corneal epithelium. The results suggest that the biological characteristics of conjunctival epithelium can be influenced by the external environment, becoming similar to those of corneal epithelium.