蝾螈视网膜色素上皮细胞向神经细胞分化:一种鼠尾视网膜再生的器官培养模型。

Yoko Ikegami, Sanae Mitsuda, M. Araki
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引用次数: 50

摘要

以蝾螈视网膜再生为实验模型,在新的培养体系下研究了视网膜色素上皮(RPE)向神经视网膜(NR)的转分化。成年蝾螈rpe与周围结缔组织(如脉络膜和巩膜)在过滤膜上进行器官培养。在体外培养第7天左右,发现带有神经突的浅色“神经元样细胞”从外植体迁移到过滤膜上。他们的人数日益增加。brdu标记研究表明,RPE细胞在体外培养条件下于第4天开始增殖,与体内视网膜再生的时间进程有时间相关性。培养的外植体的组织学观察表明,增殖的RPE细胞并没有形成在NR中常见的分层结构,而是从外植体中迁移出来。通过免疫组化检测各种神经元特异性蛋白检测神经元分化;HPC-1 (syntaxin), GABA,血清素,视紫红质和乙酰化微管蛋白。这些蛋白质的免疫反应细胞总是具有细而长的神经突样突起。大量浅着色的神经元样细胞显示HPC-1免疫反应性。成纤维细胞生长因子-2 (FGF-2)被认为是多种脊椎动物眼部组织转分化的有效因子,它以剂量依赖性的方式显著增加了神经元样细胞和hpc -1样免疫反应细胞的数量。这些结果表明,我们的培养方法保证了蝾螈RPE细胞在体外的神经分化,首次为研究蝾螈视网膜再生的组织内在因素提供了一个合适的体外实验模型系统。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Neural cell differentiation from retinal pigment epithelial cells of the newt: an organ culture model for the urodele retinal regeneration.
Transdifferentiation from retinal pigment epithelium (RPE) to neural retina (NR) was studied under a new culture system as an experimental model for newt retinal regeneration. Adult newt RPEs were organ cultured with surrounding connective tissues, such as the choroid and sclera, on a filter membrane. Around day 7 in vitro, lightly pigmented "neuron-like cells" with neuritic processes were found migrating out from the explant onto the filter membrane. Their number gradually increased day by day. BrdU-labeling study showed that RPE cells initiated to proliferate under the culture condition on day 4 in vitro, temporally correlating to the time course of retinal regeneration in vivo. Histological observations of cultured explants showed that proliferating RPE cells did not form the stratified structure typically observed in the NR but they rather migrated out from the explants. Neuronal differentiation was examined by immunohistochemical detection of various neuron-specific proteins; HPC-1 (syntaxin), GABA, serotonin, rhodopsin, and acetylated tubulin. Immunoreactive cells for these proteins always possessed fine and long neurite-like processes. Numerous lightly pigmented cells with neuron-like morphology showed HPC-1 immunoreactivity. Fibroblast growth factor-2 (FGF-2), known as a potent factor for the transdifferentiation of ocular tissues in various vertebrates, substantially increased the numbers of both neuron-like cells and HPC-1-like immunoreactive cells in a dose-dependent manner. These results indicate that our culture method ensures neural differentiation of newt RPE cells in vitro and provides, for the first time, a suitable in vitro experimental model system for studying tissue-intrinsic factors responsible for newt retinal regeneration.
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