MOLECULAR SWITCHES FOR INITIATION OF CHONDROGENESIS

描述（由申请人提供）：最近在成人骨髓腔室中发现了多能间充质干细胞。这些间充质干细胞不仅能够在体外广泛增殖，而且能够在体外分化成多种间充质细胞系，包括成骨细胞、软骨细胞、脂肪细胞、成肌细胞和间质成纤维细胞。这些间充质干细胞的体外分化再现了胚胎和成人骨骼发育过程中间充质干细胞的复杂分化过程。因此，这些多能间充质干细胞的发现为在分子水平上研究细胞命运决定提供了前所未有的机会。本研究计划的最终目的是了解间充质干细胞软骨分化起始的分子机制。为了实现这一目标，我们希望通过一种新的功能性克隆策略来确定启动间充质干细胞软骨分化的分子开关（决策基因）。具体而言，1)利用处于分化早期的人间充质细胞构建cDNA表达文库；2)利用cDNA表达文库转导小鼠间充质干细胞，筛选分化软骨细胞，克隆、测序并鉴定分化软骨细胞的cDNA插入物；3)我们将通过在间充质干细胞中单独重新表达这些基因来验证它们的调控功能。鉴定控制间充质干细胞成软骨分化的关键调控基因将有助于阐明复杂的成软骨过程的分子机制，并将揭示胚胎细胞命运的决定、骨骼模式的形成和软骨的发育。识别这些关键的调控基因也可能为开发基于干细胞的治疗慢性退行性疾病（如骨关节炎）和急性软骨损伤（如面部、脊柱和关节损伤）的疗法提供机会。

DESCRIPTION (provided by applicant): Multipotent mesenchymal stem cells are recently discovered to be present in the adult bone marrow compartment. These mesenchymal stem cells are not only able to proliferate extensively in vitro, but also are able to differentiate into several mesenchymal cell lineages in vitro, including osteoblasts, chondrocytes, adipocytes, myoblasts and stromal fibroblasts. In vitro differentiation of these mesenchymal stem cells recapitulates complex differentiation processes of mesenchymal stem cells during embryonic and adult skeletal development. Thus discovery of these multipotent mesenchymal stem cells provides unprecedented opportunities to study cell fate determination at the molecular level. The ultimate goal of this research proposal is to understand the molecular mechanism of initiation of chondrogenic differentiation of mesenchymal stem cells. To achieve this, we would like to identify molecular switches (decision-making genes) that initiate chondrogenic differentiation of mesenchymal stem cells by a novel functional cloning strategy. Specifically, 1) we will construct a cDNA expression library using human mesenchymal cells at early stages of their differentiation; 2) we will transduce mouse mesenchymal stem cells with the cDNA expression library, screen for differentiated chondrocytes, clone, sequence and identify cDNA inserts from the differentiated chondrocytes; 3) we will validate regulatory function of the identified genes by re-expression of them individually in mesenchymal stem cells. Identification of key regulatory genes controlling chondrogenic differentiation of mesenchymal stem cells will help to elucidate the molecular mechanism underlying the complex process of chondrogenesis and will shed light on embryonic cell fate determination, skeletal pattern formation and cartilage development. Identification of such key regulatory genes may also provide opportunities to develop stem cell based therapies for treatment of chronic degenerative diseases such as osteoarthritis and acute cartilage damages such as facial, spine and joint injuries.