Study on the cellular and molecular mechanisms of transdifferentiation in the neural retina regeneration

在本研究中，我们利用细胞和分子标记物分析了(1)视网膜色素上皮(RPE)细胞的转分化过程，以及(2)通过操纵分子功能分析了RPE细胞的转分化过程。结果：(1)我们将视网膜再生的过程概括为九个形态阶段，发现RPE细胞的转分化没有明显的去分化状态。(2)Pax6、Chx10-1、Notch信号分子、和神经生成素：我们证明了(I)Notch信号参与了视网膜再生过程中的神经元分化，(Ii)成年Newt RPE细胞表达神经发生的正负调控因子、神经生成素1和Hes-1基因。(Iii)我们尝试了将表达载体显微注射到活体动物眼睛的RPE细胞中，但我们仍在继续寻找更好的条件，因为注射本身往往会导致细胞死亡。利用RPE细胞无血清培养系统对FGF2的功能进行分析：我们发现：(I)FGF2在体外促进RPE细胞的去分化、增殖和泛视网膜神经元标志物的表达，(Ii)RPE细胞的转分化可能是通过MEK途径诱导的，(Iii)该途径不太可能与胚胎/幼虫途径(FGF2/FGFR-2/MEK/Pax6途径)相同，以及(Iv)胚胎/幼虫途径可能在诱导后10天内重建。

In this study, as a ground-to elucidate the cellular and molecular mechanisms of retinal regeneration in the adult newt, we analyzed (1) the transdifferentiation process of the retinal pigment epithelium (RPE) cells using cellular and molecular markers and (2) the underlying molecular pathway by manipulating molecular functions.Results of (1): we outlined the process of retinal regeneration as nine morphological stages and found that the transdifferentiation of RPE cells have no obvious dedifferentiation state.Results of (2) Functional analyses of Pax6, Chx10-1, Notch signaling molecules, and neurogenin: we demonstrated that (I) Notch signaling is involved in neuronal differentiation during retinal regeneration and (ii) the adult newt RPE cells express both positive and negative regulators of neurogenesis, neurogenin 1 and Hes-1 genes. (iii) We tried microinjection of expression vectors into RPE cells in the eye of living animal, but we still continue to find better conditions because injection itself frequently resulted in cell death. (iv) We found that defect of Musashi 1 causes retinal degeneration.Functional analyses of FGF2 using serum-free culture systems of RPE cells: we found that (I) FGF2 promotes dedifferentiation of RPE cells, proliferation and expression of pan-retinal neuron markers in vitro, (ii) transdifferentiation of RPE cells may be induced through an MEK pathway, (iii) this pathway is unlikely to be the same as embryonic/larval one (FGF2/FGFR-2/MEK/Pax6 pathway), and (iv) the embryonic/larval pathway may be reestablished within 10 days of induction.