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Molecular analysis of common mechanisms that are crucial for maintenance of hematopoietic stem cells and male germ stem cells.

对维持造血干细胞和雄性生殖干细胞至关重要的常见机制进行分子分析。

基本信息

批准号：
14370306
负责人：
OHBO Kazuyuki
金额：
$ 5.57万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

The common properties such as self-renewing and necessity of supporting cells called niche are maintained by either identical or similar molecular mechanisms. Among those common properties, we are interested in interacting molecules between stem cells and niches. Therefore, we decided to focus on cell surface molecules and secreting molecules that are crucial for interaction between stem cells and supporting cells.We generated cDNA library from purified male germ stem cells of Oct-4/GFP mice that carry a transgene expressing EGFP protein under the control of Oct-4 promoter and enhancer. We cloned genes encoding cell surface molecules and secreting molecules by a signal sequence trap method. Then, we chose the molecules that were not expressed in somatic cells, Serotoli cells and NIH3T3 fibroblasts. As consequence, we decided to focus on two molecules, mHE-4 and JAM-4. mHE-4 is one of WAP type serine proteinase inhibitor proteins and JAM-4 is a immunoglobulin type cell-adhesion molecule … More . Then, we observe the expression of those molecules by RT-PCR, northen blotting analysis and immunohistochemical analysis. Both of them were expressed in CD34-negative hematopoietic stem cells. CD34-positive hematopoietic stem cells expressed both molecules but weakly. The progenitor cells express neither mHE-4 nor JAM-4. In addition, immunohistochemical analysis showed JAM-4 was expressed in liver and kidney, and HE-4 was expressed in lung and kidney. Proteinases and their inhibitors play roles at many biological reactions. Because serine proteinase functions in digestion of bacreia, mHE may function at the process of bacterial infection. In testis, mHE-4 expressed on the cell surface of spermatogonia. Histochemical analysis of JAM-4 showed the co-localization with tight junction protein, ZO-1, suggesting JAM-4 may function as a tight junction protein. To analyze the function of those molecules in vivo, we are currently generating knock-out mice of those molecules and have already obtained the chimera mice. Less

共同的属性，如自我更新和支持细胞的必要性称为生态位，由相同或相似的分子机制维持。在这些共同的性质中，我们感兴趣的是干细胞和壁龛之间的相互作用分子。因此，我们决定将重点放在干细胞和支持细胞之间相互作用的细胞表面分子和分泌分子上。我们从OCT-4/GFP小鼠纯化的雄性生殖细胞中构建了在OCT-4启动子和增强子控制下表达EGFP蛋白的转基因c DNA文库。我们用信号序列捕捉法克隆了编码细胞表面分子和分泌分子的基因。然后，我们选择了在体细胞、Serotoli细胞和NIH3T3成纤维细胞中不表达的分子。因此，我们决定重点研究两个分子，MHE-4和JAM-4。MHE-4是一种WAP型丝氨酸蛋白酶抑制蛋白，JAM-4是一种免疫球蛋白型细胞黏附分子…更多。通过RT-PCR、Northern印迹分析和免疫组织化学方法观察这些分子的表达情况。二者均在CD34阴性的造血干细胞中表达。CD34阳性的造血干细胞表达这两种分子，但表达较弱。祖细胞既不表达MHE-4，也不表达JAM-4。免疫组织化学分析显示，JAM-4在肝脏和肾脏有表达，HE-4在肺和肾脏有表达。蛋白水解酶及其抑制物在许多生物反应中发挥作用。因为丝氨酸蛋白酶在细菌的消化过程中起作用，所以MHE可能在细菌感染过程中起作用。在睾丸中，MHE-4表达于精原细胞表面。组织化学分析显示JAM-4与紧密连接蛋白ZO-1共定位，提示JAM-4可能是紧密连接蛋白。为了分析这些分子在体内的功能，我们目前正在制造这些分子的敲除小鼠，并已经获得了嵌合体小鼠。较少