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

这些共同的特性，如自我更新和需要支持细胞（称为小生境），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细胞表面分子和分泌分子的干细胞和支持细胞之间的相互作用是至关重要的，我们从纯化的Oct-4/GFP小鼠的雄性生殖干细胞，携带转基因表达EGFP蛋白的Oct-4启动子和增强子的控制下，构建cDNA文库。我们用信号序列捕获法克隆了编码细胞表面分子和分泌分子的基因。然后，我们选择在体细胞、Serotoli细胞和NIH 3 T3成纤维细胞中不表达的分子。因此，我们决定专注于两种分子，mHE-4和JAM-4。mHE-4是WAP型丝氨酸蛋白酶抑制剂蛋白，JAM-4是免疫球蛋白型细胞粘附分子 关于我们 .通过RT-PCR、Northen blotting和免疫组化等方法观察这些分子的表达。两者均在CD 34阴性造血干细胞中表达。CD 34阳性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。免疫组化结果显示，JAM-4在肝、肾中表达，HE-4在肺、肾中表达。蛋白酶及其抑制剂在许多生物反应中发挥作用。由于丝氨酸蛋白酶在细菌的消化中起作用，因此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，表明JAM-4可能是一种紧密连接蛋白。为了在体内分析这些分子的功能，我们目前正在产生这些分子的敲除小鼠，并已获得嵌合小鼠。少

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