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Morphological analysis of mechanisms for organ remodeling using amphibian digestive tract as a model

以两栖动物消化道为模型的器官重塑机制的形态学分析

基本信息

批准号：
17570051
负责人：
OKA Atsuko
金额：
$ 2.42万
依托单位：
Nippon Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17570051/
关键词：
digestive tract organ remodeling tissue interaction stem cell transgenic frog metamorphosis thyroid hormone response gene organ culture 甲状腺ホルモン受容体アフリカツメガエルアポトーシス遺伝子導入免疫組織化学

项目摘要

In the Xenopus laevis intestine during metamorphosis, by the inductive action thyroid hormone (TH), the larval epithelium undergoes apoptosis, whereas stem cells appear, actively proliferate and form the adult epithelium similar to the mammalian one. In this study, we studied molecular mechanisms of the larval-to-adult intestinal remodeling as a model for mammalian organ regeneration, using culture and immunohistochemical procedures with transgenic frogs. Our results obtained are summarized as follows.1. Using transgenic tadpoles expressing GFP under the control of CMV promoter, we showed that intestines isolated from the transgenic tadpoles continued to express GFP in vitro during TH-induced remodeling, but wild intestines did not. Then, to clarify the tissue origin of the stem cells, we performed epithelial-connective tissue recombination experiments with the wild and transgenic intestines and examined GFP expression of the stem cells, which can be identified in the cultured explants with immunohistochemical markers such as P-PTEN and Akt. Our results suggest the epithelial origin of the stem cells.2. To investigate roles of epithelial-connective tissue interactions in the intestinal remodeling, we used intestines isolated from transgenic tadpoles expressing dominant positive TH receptor (dpTR), which can activate TR responsive promoters constitutively under the control of a heat shock-inducible promoter. We performed epithelial-connective tissue recombination experiments with the wild and transgenic intestines and cultured the explants in the absence of TH. Transgenic expression of dpTR in the epithelium alone induced apoptosis of the larval epithelium, but did not lead to development of the adult epithelium originating from the stem cells. This implicates that TH response genes expressed in the connective tissue are essential for the control of the stem cells. Thus, the Xenopus intestinal model is useful for clarifying molecular aspects of the stem cell niche.

在非洲爪蟾肠变态过程中，在甲状腺激素（TH）的诱导作用下，幼虫上皮发生凋亡，而干细胞出现并积极增殖，形成与哺乳动物上皮相似的成体上皮。在这项研究中，我们利用转基因青蛙的培养和免疫组织化学方法，研究了作为哺乳动物器官再生模型的幼虫到成虫肠道重塑的分子机制。我们的研究结果总结如下：使用CMV启动子控制下表达GFP的转基因蝌蚪，我们发现从转基因蝌蚪分离的肠道在th诱导的体外重塑过程中继续表达GFP，而野生肠道则没有。然后，为了明确干细胞的组织来源，我们用野生和转基因肠道进行了上皮结缔组织重组实验，并检测了干细胞的GFP表达，这可以在培养的外植体中用免疫组织化学标记（如P-PTEN和Akt）识别。我们的结果提示了干细胞的上皮起源。为了研究上皮-结缔组织相互作用在肠道重塑中的作用，我们使用了从表达显性TH受体（dpTR）的转基因蝌蚪分离的肠道，dpTR可以在热休克诱导启动子的控制下组成性地激活TR响应启动子。我们用野生和转基因肠道进行了上皮结缔组织重组实验，并在不含TH的情况下培养了外植体。在上皮中单独表达dpTR转基因基因可诱导幼虫上皮细胞凋亡，但不能诱导干细胞形成成体上皮细胞。这表明结缔组织中表达的TH反应基因对干细胞的控制至关重要。因此，非洲爪蟾肠道模型有助于阐明干细胞生态位的分子方面。