Hyuga M, Kodama R, Eguchi G
Department of Molecular Biomechanics, School of Life Science, Graduate University for Advanced Studies, Okazaki, Japan.
Int J Dev Biol. 1993 Jun;37(2):319-26.
In vitro transdifferentiation of retinal pigmented epithelial cells of the chick embryo into lens cells can be markedly enhanced by culture in the presence of testicular hyaluronidase and phenylthiourea. Since the commercial preparations of hyaluronidase that had previously been used were very crude, a search for the actual effective molecule(s) enhancing lens transdifferentiation was conducted. First, we purified the enzyme and tested the effect of the purified hyaluronidase. Highly purified hyaluronidase itself did not enhance lens transdifferentiation. The crude hyaluronidase was then separated according to affinity with heparin, considering the possibility that the fibroblast growth factor (FGF) is contained in the crude hyaluronidase. Transdifferentiation-enhancing activity was detected in the fraction which was bound to heparin and eluted with 2 M NaCl, where no hyaluronate-degrading activity existed. Analysis of the fraction by SDS-PAGE revealed the existence of an 18 kDa protein whose NH2-terminal sequence was identical to that of basic FGF. The basic FGF derived from bovine brain also enhanced lens transdifferentiation of pigmented epithelial cells. These findings suggest that basic FGF must play a major role in enhancing transdifferentiation of pigmented epithelial cells to lens cells.
在睾丸透明质酸酶和苯硫脲存在的情况下进行培养，鸡胚视网膜色素上皮细胞向晶状体细胞的体外转分化可得到显著增强。由于之前使用的商业透明质酸酶制剂非常粗糙，因此开展了对增强晶状体转分化的实际有效分子的探寻。首先，我们对该酶进行了纯化，并测试了纯化后的透明质酸酶的效果。高度纯化的透明质酸酶本身并不能增强晶状体转分化。考虑到粗制透明质酸酶中可能含有成纤维细胞生长因子（FGF），随后根据与肝素的亲和力对粗制透明质酸酶进行了分离。在与肝素结合并用2M氯化钠洗脱的级分中检测到了转分化增强活性，该级分不存在透明质酸降解活性。通过SDS-PAGE对该级分进行分析，发现存在一种18kDa的蛋白质，其氨基末端序列与碱性FGF的相同。源自牛脑的碱性FGF也增强了色素上皮细胞的晶状体转分化。这些发现表明，碱性FGF在增强色素上皮细胞向晶状体细胞的转分化过程中必定起主要作用。
