Analysi of molecular mechanisms of tissue reparative regeneration through transdifferentiation

1)我们建立了一种独特的鸡胚色素上皮细胞（佩奇）培养体系，用于研究转分化的细胞和分子机制。通过该系统，可以产生多能去分化细胞（dePEC）的纯的和大的群体，并且可以人工调节从佩奇到透镜表型的转分化的每个过程。清楚地证明了这些dePEC分化为透镜或色素细胞的双能性质。2）使用该系统，我们表征了多能dePEC的性质，并证明了这些dePEC通过约15个有丝分裂周期维持其分化为透镜或色素细胞的双能性质，然后逐渐失去其分化色素细胞表型的潜力。3）成功制备了蝾螈透镜特异性晶体蛋白单克隆抗体。将这些抗体应用于scleencDNA文库，我们克隆了一个主要的胚晶状体<gamma>蛋白亚类基因（命名为<gamma>A1），并测定了其全碱基序列。结果表明，A1基因<gamma>在蝾螈透镜细胞中特异性表达，而在正常成年蝾螈虹膜色素上皮细胞中不表达。4）我们成功制备了与蝾螈细胞表面或细胞间分子发生交叉反应的特异性单克隆抗体。在晶状体切除后不久，发现抗原分子仅在再生透镜的虹膜背缘迅速衰减，这表明这些抗原分子在再生的调节中起着重要作用。通过本一般性研究获得的这些结果应该是深入理解再生调节机制的基础信息。

1) We established a unique cell culture system of chick embryonic pigmented epithelial cells (PECs) for studying cellular and molecular mechanisms of transdifferentiation. By this system a pure and large population of multipotent dedifferentiated cells (dePECs) can be produced and each process of transdifferentiation from PECs to lens phenotypes can be artificially regulated. The bipotent nature of these dePECs for differentiating to either lens or pigment cells was clearly demonstrated.2) Using this system we characterized the nature of multipotent dePECs and demonstrated that these dePECs maintained their bipotent nature differentiating to either lens or pigment cells through about 15 mitotic cycles and then gradually lost their potential for differentiating pigment cell phenotypes. The mode of expressin of differentiative potentials in dePECs must be closely related to their aging.3) We succeeded in production of monoclonal antibodies against newt lens specific crystallins. Applying these antibodies to scleen cDNA library, we cloned a gene for the major enbryonic <gamma> -crystallin subclass (named <gamma> A1) and determined its whole base sequence. It was demonstrated that the gene for <gamma> A1 was specifically expressed in newt lens cells but not in pigmented epithelial cells of the normal adult newt iris.4) We succeeded in production of unique monoclonal antibodies crossreacting with newt cell surface or intercellular molecules which widely distributed in many types of newt tissues including dorsal and ventral iris epithelia. The antigen molecules was found to decay rapidly in only the dorsal marginal iris, from which a lens was regenerated, soon after lentectomy, suggesting the essential roles of these antigen molecules in regulation of regeneration.These results obtained through this General Research should be the fundamental information for deeply understanding the regulatory mechanism of regeneration.