Molecular Mechanisms of Transdifferentiation and Stabilization in Differentiation of Animal Tissue Cells

1996年进行的研究计划取得了以下成果。(1)通过对8 ~ 10日龄鸡胚视网膜色素上皮细胞（retinal pigmented epithelial cells，佩奇）体外转分化体系的研究，发现bFGF和bFGF都是调控佩奇转分化的重要因子。此外，通过将这两种生长因子引入细胞培养，我们成功地建立了一个新的和高度稳定的体外实验系统的虹膜佩奇从初生小鸡。该系统使我们能够在不使用任何人工试剂的情况下操纵佩奇的透镜转分化的整个过程。(2)利用该系统的分析表明，MAP激酶的激活是佩奇去分化的必要条件，并且两个同源盒基因pax 6和six 3的表达通过bFGF和EGF的协调功能而显著增强。(3)通过对佩奇转分化过程中细胞外基质（ECM）变化的详细分析，获得了以下结果：β 1整联蛋白通过将信号从ECM组分传递到佩奇，在佩奇分化状态的稳定维持中起重要作用，并且当佩奇去分化开始时，β 1整联蛋白分子容易被磷酸化而失去其功能。(4)通过对禽小眼症基因（mi）的克隆和结构功能分析，发现mi突变与鹌鹑银突变体色素上皮异位形成神经视网膜有关，mi是脊椎动物PEC脱分化和转分化的必需基因之一。(5)这些发现必须提供基本的信息，接近所需的分子机制，参与转分化和稳定的组织细胞分化一般。

The following results were obtained by conducting the research plans in 1996.(1) Through extensive studies to improve our authentic in vitro transdifferentiation system of retinal pigmented epithelial cells (PECs) from 8 to 10-day-old chick embryos, both bFGF were found to be essential factors regulating transdifferentiation of PECs. In addition, by introducing these tow growth factors to cell cultures, we succeeded in establishing a novel and highly stable in vitro experimental system of the iris PECs from neuwly-hatched chicks. The system allows us to manipulate the whole process of the lens transdifferentiation of PECs without any artificial reagent.(2) Analyzes using this system revealed that activation of MAP kinase is an essential requisite for dedif-ferentiation of PECs and that the expression of two homeobox genes, pax6 and six3 are dramatically enhanced by coordinated functions of bFGF and EGF.(3) The following results were obtained through detailed analysis of changes in the extracellular matrix (ECM) during transdifferentiation of PECs that beta1 integrin plays an essential role in stable maintenance of the differentiated state of PECs by transmitting signals from ECM components to PECs, and that beta1 integrin molecules are readily phospholylated to lose their function when dedifferentiation of PECs is initiated.(4) It was resulted in by cloning and structural and functional analysis of avian microphthalmia gene (mi) that mutation of mi must be responsible for ectopic formation of the neural retina from the developing pigmented epithelium in Silver mutant of the quail, and also that mi is one of essential genes, which regulate dedifferentiation and transdifferentiation of the vertebrate PEC.(5) These findings must provide the fundamental information required for approaching to the molecular mechanisms involved in transdifferentiation and stability in differentiation of tissue cells in general.