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Research to enhance potentials of mesenchymal calls by introducing Nanog gene and its application to regenerative medicine of bone and cartilage

导入Nanog基因增强间充质细胞潜能的研究及其在骨软骨再生医学中的应用

基本信息

批准号：
18592166
负责人：
HOSHI Kazuto
金额：
$ 1.93万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18592166/
关键词：
bone, cartilage regenerative medicine stem cell 骨軟骨再生医療 Nanog遺伝子分化

项目摘要

In order to apply regenerative medicine to clinical settings of oral surgery, we introduced Nanog gene, a pluripotency sustaining factor in embryonic stem cells (ES cells), into mesenchymal cells and attempted to promote their self-renewal and differentiation potentials. The purpose of the present study was to enhance the differentiation potential of mesenchymal stem cells by introducing Nanog gene and to realize the efficient formation of bone or cartilage. First of all, Nanog gene was transduced into some established cell lines and its function was analyzed. When Nanog expression was examined at gene and protein level by PCR and Western blotting, it was almost undetectable in mice cell lines, C3H10T1/2, MC3T3-E1 and ATDC5. Then, we investigated the cell differentiation of above-mentioned cells into which Nanog cDNA was introduced by viral vectors in a temporal or constitutive manner. The results suggested that Nanog may control the osteogenic differentiation. For gene silencing experiments using Nanog RNAi, virus-mediated siRNA delivery was conducted to verify its effect on cell differentiation and proliferation. Furthermore, the cells were cloned to investigate the silencing effects of Nanog. Finally, we examined the molecular mechanisms of osteogenic differentiation in the cells under constitutive expression of Nanog. Western blotting analysis revealed phosphorylation of Smad1/5/8, which are downstream effectors in BMP signaling, was sustained in response to the introduction of Nanog gene. As to phosphorilation of STAT3, which is important for self-renewal of ES cells, it was shown by western blotting to decrease in Nanog-transduced cells. Taken together, Nanog was suggested to have important functions in controlling osteogenic/chondrogenic differentiation as well as in ES cells.

为了将再生医学应用于口腔外科的临床环境，我们将胚胎干细胞(ES细胞)中的多能支持因子Nanog基因导入间充质细胞，并试图促进其自我更新和分化。本研究的目的是通过导入Nanog基因来增强间充质干细胞的分化潜能，实现高效的骨或软骨的形成。首先，将Nanog基因导入一些已建立的细胞系，并对其功能进行分析。当用聚合酶链式反应和蛋白质印迹法检测Nanog在基因和蛋白水平上的表达时，在小鼠细胞系C3H10T1/2、MC3T3-E1和ATDC5中几乎检测不到它的表达。然后，我们研究了用病毒载体以时间或结构性的方式将Nanog cDNA导入上述细胞后的细胞分化情况。结果提示，Nanog对成骨分化有一定的控制作用。在使用Nanog RNAi的基因沉默实验中，通过病毒介导的siRNA传递来验证其对细胞分化和增殖的影响。此外，还克隆了细胞以研究Nanog的沉默作用。最后，我们研究了在Nanog结构性表达下细胞成骨分化的分子机制。Western blotting分析表明，在BMP信号转导过程中的下游效应分子Smad1/5/8的磷酸化持续受到Nanog基因的影响。至于对ES细胞自我更新起重要作用的STAT3的磷酸化，Western blotting显示在Nanog转导的细胞中STAT3的磷酸化减少。综上所述，Nanog不仅在ES细胞中具有重要的功能，而且在成骨/软骨分化中也具有重要作用。