Identification of molecules determining the stromal-cell dependent hematopoiesis

我们已经从SV-40T抗原转基因小鼠身上建立了33个骨髓基质细胞系。在这些克隆中，27个克隆支持红系集落形成，6个克隆不支持。为了确定决定红系集落形成活性的分子，我们用DNA微阵列比较了支持红系生成的细胞系(E+；TBR9,184，31-2)和不支持红系集落形成的细胞系(E-；TBR17，33,511)的基因表达谱。在差异表达的基因中，我们选择了上调的基因TN-C(TN-C)作为最后一个项目的候选基因，并证明了它在基质细胞依赖的红细胞生成中发挥了重要作用(Exp hematol 34：519-527,2006)。在本项目中，我们从其他候选基因中选择了2810021G24和Delta-like 1，并对这两个分子的功能进行了检测，2810021G24在E+细胞中的表达是E-细胞中的14.0倍。2810021G24小干扰RNA(SiRNA)存在时，TBR9的红系集落数显著低于对照siRNA，提示2810021G24也参与了基质细胞依赖性的红细胞生成。为了证实这一假设，我们试图建立2810021G24基因敲除小鼠，但没有成功，E细胞系中Delta-like 1的表达是E+细胞系的20.0倍。然而，Delta样1siRNA的加入并没有导致TBR17中红系集落数量的增加，这表明Delta样1不是E基质细胞系中基质细胞依赖性红系生成的唯一抑制因子。

We have previously established 33 bone marrow stromal cell lines from SV 40 T-antigen transgenic mice. Of these, 27 clones supported erythroid colony formation, while 6 clones did not. To identify the molecules which determine the erythroid colony forming activities, we compared the gene expression profiling by DNA microarray between cell lines which support erythropoiesis (E+; TBR9, 184, 31-2) and cell lines which do not (E-; TBR17, 33, 511). Among the differentially expressed genes, we selected one of the upregulated genes, tenascin-C (TN-C), as a candidate in the last project, and demonstrated that it plays an important role in the stromal cell-dependent erythropoiesis (Exp Hematol 34:519-527, 2006). In this project, we have selected 2810021G24 and delta-like 1 among other candidate genes, and examined the function of these two molecules.The expressions of 2810021G24 in E+ cell lines were 14.0 times higher than in E- cell lines. The number of erythroid colonies in the presence of 2810021G24 small interfering RNA (siRNA) was significantly lower than that of control siRNA in TBR9, suggesting that 2810021G24 is also involved in the stromal-cell dependent erythropoiesis. To confirm this hypothesis, we have attempted to establish knockout mice of 2810021G24, but it was not successful.The expressions of delta-like 1 in E- cell lines were 20.0 time higher than E+ cell lines. However, the addition of delta-like 1 siRNA did not result in the increase of the number of erythroid colonies in TBR17, suggesting that delta-like 1 is not the exclusive inhibitory factor of stromal-cell dependent erythropoiesis in the E- stromal cell lines.