Identification of molecules determining the stromal-cell dependent hematopoiesis

我们已经从SV40-T抗原转基因小鼠中建立了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。本研究的目的是确定决定这些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集落形成的细胞株(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，我们选择了候选基因，结合定量RT-PCR的结果，检测了小干扰RNA(SiRNA)和外源蛋白对红系集落形成的影响，在所检测的7226个基因中，有138个基因在E+中上调或下调了3倍以上，282个基因在E+中上调或下调了3倍以上。我们选择了上调基因Tn-C(TN-C)作为候选基因。TN-C在E+细胞中的表达均高于三种E-细胞系，平均值为3.6倍。在TN-C siRNA存在的情况下，TBR9和TBR184的红细胞集落数分别为20.7±6.3和13.3±5.3，显著低于对照siRNA的4.7±4.8和0.3±0.5(p=0.02)。此外，外源性TN-C的加入增加了TBR184(13.3±3.5比20.0±2.0；p=0.04)和TBR31-2(7.5±3.1比13.5±2.6；p=0.03)的红细胞集落数。这些结果表明TN-C参与了基质细胞依赖的红细胞生成。

We have previously established 33 bone marrow stromal cell lines from SV40-T antigen transgenic mice. Of these, 27 clones supported erythroid colony formation, while 6 clones did not. The objective of this study is to identify the molecules which determine these erythroid colony forming activities.We compared the gene expression profiling by DNA microarray between cell lines which support eaythmpoiesis (E+;TBR9,184,31-2) and cell lines which do not (E-; TBR17,33,511). Among the differentially expressed genes, we selected candidate genes, with the results of quantitative RT-PCR, examined the effect of small interfering RNA (siRNA) and the addition of exogenous proteins on the erythroid colony formation.Out of 7226 genes examined, 138 and 282 genes were upregulated and downregulated in E+ by 3-fold or more, respectively. We have selected one of the upregulated genes, tenascin-C (TN-C), as a candidate. The expressions of TN-C in E+ were all higher than the three E- cell lines with a mean of 3.6-fold. The number of erythroad colonies in the presence of TN-C siRNA was significantly lower than that of control siRNA in TBR9 (20.7±6.3 versus 4.7±4.8 colonies ; p=0.01) and in TBR184 (13.3±5.3 versus 0.3±0.5;p=0.02). Moreover, the addition of exogenous TN-C enhanced the number of erythmid colonies in TBR184 (13.3±3.5 versus 20.0±2.0;p=0.04) and in TBR31-2 (7.5±3.1 versus 13.5±2.6;p=0.03).These results suggest that TN-C is responsible for detemining the stromal cell-dependent erythropoiesis.