Analysi of molecular mechanisms of tissue reparative regeneration through transdifferentiation

1)建立了鸡胚色素上皮细胞（PECs）独特的细胞培养体系，研究其转分化的细胞和分子机制。通过该系统可以产生大量的多能去分化细胞（deecs），并且可以人工调节从多能去分化细胞到晶状体表型的每个转分化过程。这些depec分化为晶状体细胞或色素细胞的双能性被清楚地证明。2)利用该系统表征了多能性depec的性质，并证明这些depec在15个有丝分裂周期中保持了向晶状体或色素细胞分化的双能性，然后逐渐失去了分化色素细胞表型的潜力。depec中微分电位的表达方式必然与衰老密切相关。3)成功制备了针对蝾螈晶状体特异性结晶蛋白的单克隆抗体。利用这些抗体筛选cDNA文库，我们克隆了一个主要胚胎< γ > -晶体蛋白亚类的基因（命名为< γ > A1），并确定了其全碱基序列。结果表明，< γ > A1基因在蝾螈晶状体细胞中特异性表达，而在正常成年蝾螈虹膜色素上皮细胞中不表达。4)成功制备了与蝾螈细胞表面或细胞间分子交叉反应的独特单克隆抗体，该单克隆抗体广泛分布于包括背、腹侧虹膜上皮在内的多种蝾螈组织中。发现抗原分子在晶状体切除后仅在晶状体再生的背缘虹膜中迅速衰减，提示这些抗原分子在晶状体再生的调节中起重要作用。这些研究结果为深入了解再生调控机制提供了基础信息。

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.