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

在本研究中，作为阐明成年蝾螈视网膜再生的细胞和分子机制的基础，我们分析了（1）使用细胞和分子标记的视网膜色素上皮（RPE）细胞的转分化过程和（2）通过操纵分子功能的潜在分子途径。我们将视网膜再生过程概括为9个形态学阶段，发现RPE细胞转分化没有明显的去分化状态。（2）Pax 6、Chx 10 -1、Notch信号分子的功能分析结果，和neurogenin：我们证明了（I）Notch信号传导参与视网膜再生过程中的神经元分化，和（ii）成年蝾螈RPE细胞表达神经发生的正和负调节因子，神经生成素1和Hes-1基因。(iii)我们尝试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，因为注射本身经常导致细胞死亡。(iv)我们发现Musashi 1的缺陷导致视网膜变性。使用RPE细胞的无血清培养系统的FGF 2的功能分析：我们发现（I）FGF 2在体外促进RPE细胞的去分化、增殖和全视网膜神经元标记物的表达，（ii）RPE细胞的转分化可通过MEK途径诱导，（iii）该途径不太可能与胚胎/幼虫途径（FGF 2/FGFR-2/MEK/Pax 6途径）相同，和（iv）胚胎/幼虫途径可以在诱导后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.