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

共同的特性，如自我更新和需要支持细胞称为生态位是由相同或类似的分子机制维持。在这些共同特性中，我们对干细胞和壁龛之间的相互作用分子感兴趣。因此，我们决定将重点放在细胞表面分子和分泌分子上，这些分子对干细胞和支持细胞之间的相互作用至关重要。我们从纯化的Oct-4/GFP小鼠雄性生殖干细胞中获得cDNA文库，该细胞在Oct-4启动子和增强子的控制下携带表达EGFP蛋白的转基因基因。利用信号序列诱捕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、血清细胞和NIH3T3成纤维细胞中不表达的分子。因此，我们决定把重点放在两个分子上，mHE-4和JAM-4。mHE-4是WAP型丝氨酸蛋白酶抑制蛋白之一，JAM-4是免疫球蛋白型细胞粘附分子。然后通过RT-PCR、northern blotting和免疫组化分析观察这些分子的表达情况。两者均在cd34阴性造血干细胞中表达。cd34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。免疫组化分析显示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。由于丝氨酸蛋白酶在细菌的消化中起作用，mHE可能在细菌感染过程中起作用。在睾丸中，mHE-4在精原细胞表面表达。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可能具有紧密连接蛋白的功能。为了分析这些分子在体内的功能，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。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