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

在发生在纵骨的软骨内骨化中，来自间充质细胞的软骨组织被骨化中心内的骨取代。因此，软骨内骨化先于成骨细胞骨化。因此，阐明软骨形成的分子机制是更好地理解骨形成的前提。我们已经证明，Notch信号刺激成骨细胞分化。目前的研究项目旨在阐明Notch1调控骨形成的机制。Notch1从小鼠胚胎前肢的间质凝聚阶段开始定位。有趣的是，虽然在增殖的软骨细胞中没有检测到定位，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一种活性形式的Notch(NIC)转移到细胞系中。关于ch…的辨析更多的软骨细胞受到抑制。我们利用肢芽微团培养(LBMC)进行进一步的分析。LBMC是一个已建立的模型，概括了间充质凝聚和软骨细胞分化。RT-PCR显示Notch及其相关基因在细胞凝聚和结节形成的第1天和第5天均有表达。免疫组织化学实验表明，Notch1的表达最初定位于结节内，随着细胞分化的进行，Notch1的表达转移到结节周围。为了分析Notch信号在培养体系中的作用，我们使用了一种伽马分泌酶抑制剂DAPT来阻断Notch信号。DAPT阻断Notch信号明显促进结节软骨前凝聚和融合的启动，这种作用可被Notch胞质结构域的外源表达逆转。这些结果表明，Notch信号可能通过负调控软骨形成前凝聚和结节的形成而在软骨细胞分化中发挥重要作用。我们利用逆转录病毒介导的信号序列陷阱(SST-REX)方法，从软骨形成Lell系(ATDC5)凝聚阶段的mRNA组分中构建了一个cDNA文库。通过对5.7×10^3克隆的筛选，获得了486个不依赖IL-3的克隆。对克隆进行DNA测序分析，鉴定出编码157个已知蛋白和4个新蛋白的基因。一系列的分析表明，SST-REX方法是一个有用的实验系统，可以识别参与复杂的骨形成机制的基因。较少

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