Development of bone regeneration therapy for osteoporosis using humanized mice

目前项目的目标是利用HuBone-NOD-SCID小鼠开发骨质疏松症研究的临床前动物模型系统，重点关注以下几点：Notch对骨形成的调节和机械负荷对骨量的影响。携带Notch配体基因突变的基因工程小鼠会出现骨骼异常。这促使我们研究Notch在骨和软骨形成中的作用。我们发现成骨细胞系MC3T3- e1 （MC3T3）在分化早期表达Notch，随后表达下降。通过Notch1/腺病毒载体介导将Notch1基因（激活胞浆区：NIC）导入MC3T3细胞。将该基因引入细胞后，钙化结节的形成增加。将同一载体转染到人间充质干细胞（HMCS）中。造血干细胞中NIC的表达可诱导自发和受刺激的成骨细胞分化。将更多的HMSC用病毒载体感染后皮下植入NOD-SCID小鼠。我们证实，从小鼠身上恢复的支架中血管生成增加。NIC转染抑制HMSC中脂肪细胞的分化。长骨是通过软骨内成骨形成的。因此，研究Notch对软骨形成的影响具有重要意义。载体被转移到软骨细胞系（ATDC5），在加入胰岛素后分化成软骨细胞。NIC可抑制软骨细胞分化。我们的研究结果表明Notch促进成骨细胞的分化，但抑制间充质干细胞向脂肪细胞和软骨细胞的分化。目前，我们正在进行实验，以破译细胞分化途径所涉及的分子机制。我们研究了绝经后妇女体育锻炼与骨量之间的关系。体育锻炼被证明可以增加骨量，但在停止体育锻炼后，增加的骨量很快就会消失。少

The objective of the current project is to develop a pre-clinical animal model system for osteoporosis research using HuBone-NOD-SCID mice, with emphasis on the following points; regulation of bone formation by Notch and changes of bone mass by mechanical load. The genetically engineered mice carrying mutations in the Notch ligand genes undergo skeletal abnormalities. This prompted us to investigate involvement of Notch in bone and cartilage formation. We have found that the osteoblastic cell line, MC3T3-E1 (MC3T3), expresses Notch at early differentiation stage, then the expression declines. The Notch1 gene (activated cytoplasmic region: NIC) was introduced into MC3T3 cells mediated via Notch1/adeno virus vector. The introduction of the gene into the cells resulted in increased calcified nodule formation. The same vector was transfected into human mesenchymal stem cells (HMCS). NIC expression in HMSC induced both spontaneous and stimulated osteoblastic differentiation. Scaffold carryi … More ng HMSC were infected with the viral vector and were implanted into NOD-SCID mice subcutaneously. We confirmed increased angiogenesis in the scaffolds recovered from the mice. NIC transfection inhibited adipocyte differentiation in HMSC. Long bones are developed by endochondral ossification. Thus, it is important to investigate the effects of Notch on chondrogenesis. The vector was transferred to the chondrogenic cell line (ATDC5) which differentiates into chondrocytes following the addition of insulin. NIC was demonstrated to inhibit the chondrocyte differentiation. Our results suggest that Notch promotes osteoblastic differentiation, but inhibi the differentiation into adipocyte and chondrocyte from MSC. Currently, we are working on the experiments to decipher the molecular mechanisms involved in the cellular differentiation pathways. We have investigated the relationship between physical exercise and bone mass in postmenopausal women. Physical exercise was demonstrated to increase bone mass, but the increased bone mass was quickly lost after withdrawal from physical exercise. Less