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

通过1996年的研究计划，获得了以下结果。(1)为完善8 ~ 10日龄鸡胚胎视网膜色素上皮细胞（PECs）的体外转分化系统，我们进行了大量的研究，发现两种bFGF都是调节PECs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种新的、高度稳定的雏鸡虹膜PECs体外实验系统。该系统使我们能够在不需要任何人工试剂的情况下操作PECs晶状体转分化的整个过程。(2)利用该系统分析发现，MAP激酶的激活是PECs去分化的必要条件，并且bFGF和EGF的协同作用显著增强了pax6和six3两个同源盒基因的表达。(3)通过详细分析PECs转分化过程中细胞外基质（extracellular matrix， ECM）的变化，得到以下结果：beta1整合素通过将ECM组分的信号传递给PECs，对PECs分化状态的稳定维持起着至关重要的作用，当PECs开始去分化时，beta1整合素分子容易被磷酸化而失去功能。(4)对禽小眼基因（mi）的克隆和结构功能分析表明，该基因的突变可能是导致鹌鹑银突变体在色素上皮发育过程中神经视网膜异位形成的重要基因之一，也是调控脊椎动物视网膜去分化和转分化的重要基因之一。(5)这些发现必须为研究组织细胞转分化的分子机制和分化的稳定性提供必要的基础信息。

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.