Development of novel temperature-responsive culture dish modified with bioactive compounds for functional maintenance and proliferation of corneal epithelial cells

本研究的目的是开发一种新型的温度反应培养皿，该培养皿用生物活性化合物化学固定，并在这种新型培养皿上培养和增殖上皮干细胞，而不使用NIH3T3细胞作为饲养层。对上皮干细胞培养条件的探索性研究表明，3T3饲养层分泌的部分生物活性化合物具有功能性ECM作用（制备中的科学论文）。为了进一步探索生物活性化合物，我们以不同种类的小鼠细胞系为喂料层，通过对角膜上皮细胞集落形成和多聚行为的评价，确定了一个小鼠细胞系作为阳性对照和阴性对照。采用Taqman PCR方法，将从阳性和阴性对照细胞分离的mrna与ECM来源的30种基因进行遗传和定量比较。同样，从3T3饲养层分离的mrna也采用Taqman PCR方法（一篇正在准备的科学论文）。因此，我们成功地鉴定了几个可能与饲喂层激活有关的基因。到目前为止，我们正在细胞生物学上研究这些化合物作为候选物对角膜上皮细胞的行为，将基因产物或基因产物特异性抗体添加到培养基中。除了将产物或抗体加入培养基外，我们还尝试通过羧基修饰新型温度响应培养皿表面的生物活性化合物。我们已经发现，用RGD和胰岛素肽等生物活性化合物修饰的新型培养皿改善了内皮细胞的粘附性能和增殖。目前，我们正在推进用RGD和胰岛素肽修饰的新型培养皿培养角膜上皮细胞的条件研究。

Objectives of this research are development of novel temperature responsive culture dish, which is chemically immobilized with bioactive compounds and to culture and proliferate epithelial stem cells on the novel dishes without using NIH3T3 cells as feeder layer. Exploratory investigation of culture conditions of epithelial stem cells elucidates that a part of bioactive compounds secreted form 3T3 feeder layer functionally play a role of ECM (a scientific paper in preparation). For following exploration of the bioactive compounds, a mouse cell line as positive and negative control was determined from evaluation of behavior of colony formation and multiplayer of corneal epithelial cells, using various kinds of mouse cell lines as feeder layer. The mRNAs isolated from the positive and negative control cells were genetically and quantitatively compared with 30 species of genes derived from ECM, using Taqman PCR method. Likewise, mRNAs isolated from 3T3 feeder layers were also subjected to the Taqman PCR method (a scientific paper in preparation). Consequently, we successfully identified several geneswhich possibly relate to activation of feeder layers. Until now, we are cell-biologically investigating behaviors of the compounds as candidates against corneal epithelial cells, adding gene product or antibody specific for the gene products into the culture medium. Besides addition of the products or the antibody into the medium, we are also trying to modify the surfaces of novel temperature-responsive culture dish with the bioactive compounds via carboxyl groups. We have already found that the novel culture dished modified with bioactive compounds such as RGD and insulin peptides improved adhesion properties and proliferation of endothelial cells. Now, we are promoting investigation of culture conditions of corneal epithelial cells onto the novel culture dished modified with RGD and insulin peptides.