Molecular study of hematopoiesis-supporting ability of C3H10T1/2 mouse embryo fibroblasts

为了研究基质细胞造血支持能力的分子机制，我们使用了诱导分化的小鼠胚胎成纤维细胞系C3H10T1/2 （10T1/2）。将10T1/2细胞短暂暴露于5-氮胞苷后，建立稳定的前脂肪细胞和成肌细胞系。在适当的条件下，这些细胞系最终分别分化为脂肪细胞和肌管。造血支持能力在前脂肪细胞阶段（A54前脂肪细胞）显著提高，在脂肪细胞终末分化后降低。为了确定有助于前脂肪细胞造血支持能力的分子，我们筛选了A54前脂肪细胞中差异表达的基因，并通过mRNA差异显示法分离了新基因。该基因被定义为在脂肪细胞分化过程中下调的基因-1 （drad-1）。drad-1在其他具有造血支持能力的小鼠前脂肪细胞，即ST2和PA6细胞中表达。此外，还发现drad-1在小鼠骨髓中表达。然而，由drad-1编码的蛋白质的功能目前尚不清楚。此外，我们还研究了间质细胞分化过程中CD34 mRNA表达的变化。结果，CD34 mRNA由亲本10T1/2细胞组成表达，而不是由脂肪形成或肌形成决定的细胞表达。这一发现支持了这样一种观点，即CD34也可能是基质祖细胞的标记物，并在细胞分化为表型不同的基质元素时丢失。10t1 /2衍生细胞系可以为分析基质细胞的发育和分化以及寻找新的基质细胞衍生因子提供有价值的工具。

To investigate the molecular aspects of hematopoiesis-supporting ability of stromal cells, we used a differentiation-inducible mouse embryo fibroblast cell line, C3H10T1/2 (10T1/2). Stably determined preadipocyte and myoblast cell lines were established after a brief exposure of 10T1/2 cells to 5-azacytidine. These cell lines terminally differentiated into adipocytes and myotubes, respectively, under appropriate conditions. The hematopoiesis-supporting ability was significantly elevated at the preadipocyte stage (A54 preadipocyte), and was reduced after terminal adipocytic differentiation. To identify molecules that contribute to the hematopoiesis-supporting ability of preadipocytes, we screened genes that were differentially expressed in A54 preadipocytes and isolated the novel gene by mRNA differential display method. This gene was defined as a gene that was down-regulated during adipocyte differentiation-1 (drad-1). The drad-1 was expressed in other mouse preadipocytes, namely, ST2 and PA6 cells, that also have hematopoiesis-supporting ability. Moreover, the drad-1 was found to be expressed in mouse bone marrow. However, the function of the protein encoded by drad-1 is currently unknown. In addition, we investigated the change in CD34 mRNA expression during stromal cell differentiation. As a result, CD34 mRNA was constitutively expressed by parent 10T1/2 cells but not by adipogenically or myogenically determined cells. This finding supports the concept that CD34 may also be a marker of stromal progenitors and is lost as the cells differentiate into phenotypically distinct stromal elements. The 10T1/2-derived cell lines could provide a valuable tool to aid in the analysis of stromal cell development and differentiation and the search for novel stromal cell-derived factor (s).