Identification of molecules determining the stromal-cell dependent hematopoiesis

我们以前建立了33个骨髓基质细胞系从SV 40-T抗原转基因小鼠。其中，27个克隆支持红系集落形成，而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，我们通过DNA微阵列比较了支持造血的细胞系（E+; TBR 9，184，31-2）和不支持造血的细胞系（E-; TBR 17，33，511）的基因表达谱。从差异表达基因中筛选出候选基因，通过定量RT-PCR检测小干扰RNA（siRNA）和外源蛋白对红系细胞集落形成的影响，在检测的7226个基因中，分别有138个和282个基因在E+中表达上调3倍或下调3倍以上。我们选择了一个上调的基因，tenascin-C（TN-C），作为候选基因。E+细胞系TN-C的表达均高于E-细胞系，平均为E-细胞系的3.6倍。在TBR 9（20.7±6.3对4.7±4.8个菌落; p=0.01）和TBR 184（13.3±5.3对0.3±0.5;p=0.02）中，TN-C siRNA存在下的旁路菌落数显著低于对照siRNA。此外，外源性TN-C的加入使TBR 184（13.3± 3.5vs20.0 ±2.0;p=0.04）和TBR 31 -2（7.5± 3.1vs13.5 ±2.6;p=0.03）的红细胞集落数增加，提示TN-C可能是决定基质细胞依赖性红细胞生成的因素。

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.