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

自我更新和支持细胞（称为生态位）的必要性等共同特性是通过相同或相似的分子机制来维持的，在这些共同特性中，我们对干细胞和生态位之间的相互作用感兴趣，因此，我们决定关注细胞。表面分子和分泌分子对于干细胞和支持细胞之间的相互作用至关重要。我们从Oct-4/GFP小鼠的纯化雄性生殖干细胞中生成了cDNA文库，该小鼠携带在Oct-4启动子控制下表达EGFP蛋白的转基因，并且我们克隆了编码细胞表面分子的基因。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，我们决定关注两种分子：mHE-4 和 JAM-4。 -4是WAP型丝氨酸蛋白酶抑制剂蛋白之一，JAM-4是免疫球蛋白型细胞粘附分子……更多，我们通过RT-PCR观察这些分子的表达。印迹分析和免疫组织化学分析显示，CD34阴性造血干细胞均表达这两种分子，但祖细胞不表达mHE-4，也不表达JAM-4。 JAM-4在肝脏和肾脏中表达，HE-4在肺和肾脏中表达。蛋白酶及其抑制剂在许多生物反应中发挥作用。 mHE-4在细菌消化过程中可能在细菌感染过程中发挥作用，JAM-4的组织化学分析表明mHE-4与紧密连接蛋白ZO-1共定位。 -4可能作为紧密连接蛋白发挥作用，为了分析这些分子的体内功能，我们目前正在培育这些分子的敲除小鼠，并且已经获得了嵌合体小鼠。