Studies on molecular mechanisms involved in differentiation of chondrocyte/osteoblast by Notch

在纵骨中发生的软骨内骨化中，源自间充质细胞的软骨组织在骨化中心内被骨替代。因此，软骨内骨化先于成骨细胞骨化。因此，阐明软骨发生的分子机制是更好地理解软骨发生的前提。我们已经表明，Notch信号刺激成骨细胞的分化。Notch 1在小鼠胚胎前肢间充质致密化过程中起着重要的作用，目前的研究主要集中在Notch 1调控骨形成的机制上。有趣的是，虽然在增殖的软骨细胞中不能检测到定位，但明显的免疫反应性表明其表达保留在软骨膜区域。为了评估Notch激活的效果，我们通过腺病毒载体将Notch的活性形式（NIC）转移到细胞系中。CH的分化 ...更多信息 软骨细胞受到抑制。我们利用肢芽微团培养（LBMC）进行进一步分析。LBMC是一种已建立的模型，其概括了间充质凝聚和软骨细胞分化。RT-PCR结果显示，Notch及其相关基因在培养的第1天和第5天表达，此时细胞凝聚和结节形成开始。免疫组化实验显示Notch 1的表达最初定位于结节内，并随着细胞分化的进展转移到其周边区域。我们通过使用γ-分泌酶抑制剂DAPT来破坏Notch信号传导，以分析Notch信号传导在培养系统中的功能。通过DAPT阻断Notch信号传导明显促进结节的前软骨形成凝结和融合的开始，并且这种作用通过Notch胞质结构域的外源性表达而逆转。我们利用逆转录病毒介导表达的信号序列捕获（SST-REX）方法，从软骨形成细胞系ATDC 5的软骨形成细胞中提取软骨形成细胞的软骨形成细胞中提取软骨形成细胞的软骨形成细胞的软骨形成通过筛选5.7 x 10^3个克隆，我们获得了486个因子（IL-3）非依赖性克隆。DNA序列分析鉴定了157个已知蛋白和4个新蛋白。一系列的分析表明，SST-REX方法是一个有用的实验系统，以确定参与骨形成的复杂机制的基因。少

In endochondral ossification occurring in longitudinal bone, cartilagineous tissue derived from mesenchymal cells is replaced with bone within ossification centers. Thus, endochondral ossification precedes ossification osteoblasts. Therefore it is prerequisite to elucidate the molecular mechanisms involved in chondrogenesis for better understanding of skeltogenesis. We have shown that Notch signaling stimulates the differentiation of osteoblasts. The current research projects are aimed at elucidation of mechanisms in bone formation regulated by Notch.Notch1 was localized starting from the mesenchymal condensation stage of embryonic mouse forelimbs. Interestingly, although localization could not be detected in the proliferating chondrocytes, obvious immuno-reactivity indicating its expression was retained in the perichondral region. To assess the effect of Notch activation, we transferred an active form of Notch (NIC) into the cell line by an adenovirus vector. The differentiation of ch … More ondrocytes was inhibited. We utilized limb bud micromass culture (LBMC) for further analysis. LBMC is an established model that recapitulates mesenchymal condensation and chondrocyte differentiation. RT-PCR showed that Notch and its related genes were expressed in such cultures at day 1 and day 5, when cell condensation and nodule formation were initiated. Immuno-histochiemical experiments revealed that the expression of Notch1 was initially localized within the nodules and shifted to their peripheral region as the cell differentiation progressed. We disrupted Notch signaling by using a gamma-secretase inhibitor, DAPT, to analyze the function of Notch signaling in the culture system. Blocking Notch signaling by DAPT apparently promoted the initiation of prechondrogenic condensation and fusion of the nodules, and such an effect was reversed by exogenous expression of the Notch cytoplasmic domain. These observations imply that the Notch signal may have an important role in chondrogenic differentiation by negatively regulating the initiation of prechondrogenic condensation and nodule formation.We constructed a cDNA library from the mRNA fraction derived from a chondrogenic lell line (ATDC5) at the condensation stage by using the signal sequence trap by retrovirus mediated expression (SST-REX) method. We obtained 486 factor (IL-3)-independent clones by screening 5.7 x 10^3 clones. DNA sequencing analysis of the clones identified genes encoding 157 known proteins and 4 novel proteins. A series of the analyses demonstrate that the SST-REX method is a useful experimental system to identify genes involved in the complicated mechanisms of bone formation. Less