兔角膜上皮和基质细胞共同培养模型中的相互作用

目的探讨利用细胞生物学和工程学原理在体外培养角膜上皮和基质细胞,模拟上皮和基质细胞相互作用的生存环境,了解角膜上皮细胞和基质细胞间的作用规律。方法分别进行体外兔角膜上皮细胞和基质细胞的原代培养及传代的二维平面培养,利用特殊培养装置制作上皮细胞和基质细胞共同培养的模型,达到两种细胞胞体不混合,而其分泌的成分可以相互渗透、相互作用的目的,从而更好地观察各自细胞的生长状态。描绘角膜上皮细胞和基质细胞的生长曲线及二者在相互作用模型中的生长曲线。利用激光共聚焦显微镜观察上皮细胞和基质细胞在单独培养和三维共同培养模型中上皮细胞胞间通讯的变化特点。结果角膜上皮细胞倍增时间为3．45d,共同培养的上皮细胞倍增时间为3．30d,角膜基质细胞倍增时间为2．11d,共同培养的基质细胞倍增时间为2．32d,共同培养的上皮细胞增长与单独培养的上皮细胞相比,差异有统计学意义（P〈0．01）,共同培养的基质细胞增长与单独培养的基质细胞相比,差异有统计学意义（P〈0．01）。与角膜基质细胞共同培养的角膜上皮细胞胞间通讯明显高于单独培养的基质细胞,差异有统计学意义（U=2．691,P〈0．05）。结论共同培养的细胞模型是理想的。角膜上皮细胞在与基质细胞共同存在的条件下比单独培养下的增殖能力增强,而角膜基质细胞在与上皮细胞共同存在的条件下比单独培养的增殖能力减弱;而上皮细胞在与基质细胞共同存在的条件下,细胞间通讯增强。

The interactions of stromal-epithelial in a model of co-culture in the rabbit cornea

OBJECTIVE: The purpose of the study is to produce a reconstructed cornea including epithelia and stroma by tissue engineering. The reconstructed tissue may provide a physiologic model for the investigations of interaction between corneal epithelial cells and keratocytes. METHODS: Epithelial cells and keratocytes were isolated from rabbit corneas and cultured on plastic substrates in vitro. The co-culture model was established using a special transwell in which two different cells were separated but were able to interact each other. Histological and immunohistological studies were performed to identify the cell types. Intercellular communication of both the cultured epithelial cells in pure and in co-culture with the keratocytes was studied by laser confocal scanning microscopy. RESULTS: Population doubling time (PDT) was 3.45, 3.30, 2.11 and 2.32 d in pure corneal epithelial cells, co-culture epithelial cells, pure keratocytes and co-culture keratocytes respectively. The epithelial cells in co-culture grew quicker than those in pure (P < 0.01) and the stromal cells in co-culture grew slower than those in pure (P < 0.01). Intercellular communication of the cultured epithelial cells in co-culture were more than that in pure (U = 2.691, P < 0.05). CONCLUSIONS: The co-culture model of epithelial cell and keratocyte is feasible. Under the co-culture system the responses of cell proliferation and intercellular communication are different between epithelial cells and keratocytes.