DEVELOPMENT OF A NEW THERAPEUTC APPROCH TO BONE REGENERATION FOR GENE THERAPY

我们进行了以下实验。骨髓间充质干细胞在骨再生过程中的特征我们首先在生长中的小鼠的股骨干上做了一个直径2.2毫米的圆孔。为了研究骨髓间充质细胞在骨修复过程中的增殖活性，我们探索了BrdU的掺入。这些研究表明，在我们的实验系统中，加入BrdU的增殖间充质细胞在损伤后3天增加。骨再生过程中基因表达的变化利用上述实验模型，我们通过Northern杂交分析了骨相关基因表达模式的变化。骨损伤后第4天碱性磷酸酶基因表达增强，第5天骨钙素基因表达增加。组织化学和免疫组织化学研究表明，这些分子的表达模式相似。上述实验表明，在我们的实验模型中，间充质干细胞在损伤后4天和5天出现。我们目前正在利用CDNA芯片探索与间充质干细胞相关的基因。为了探索新的治疗方法，我们用载体(PGS)将皮肤成纤维细胞移植到生长期小鼠颅骨(直径2.2 mm)缺损处。高表达BMP-2CDNA的成纤维细胞移植比表达空载体的成纤维细胞更能促进骨再生。相比之下，高表达CBFA L基因的成纤维细胞与表达空载体的成纤维细胞相比，未能刺激骨再生。

We conducted the following experiments.1. Characterization of mesenchymal stem cells during bone regenerationWe first made a round hole, 2.2 mm in a diameter, at diaphysis offemurs of growing mice. To investigate the proliferation activity of mesenchymal cells during the bone repair, we explored incorporation of BrdU. These studies demonstrated that proliferating mesenchymal cells incorporating BrdU increased 3 days after injury in our experimental system.2. Changes in gene expression during bone regenerationUsing above described experimental model, we investigated the changes in expression patterns of bone-related genes by Northern blot analysis. ALP mRNA increased on day 4 and osteocalcin mRNA increased on day 5 after bone injury. Histochemical and immunohistochemical studies indicated similar expression patterns of these molecules.3. Above experiments suggested that mesenchymal stem cells appeared during days 4 and 5 after injury in our experimental model. We are now exploring the genes related to mesenchymal stem cells by CDNA chips.4. To develop new therapeutic method, we transplanted skin fibroblasts in bone defects (2.2 mm in a diameter) in calvariae of growing mice with carrier (PGS). Transplantation of the fibroblasts over-expressing BMP-2 CDNA induced more accelerated bone regeneration than that transplanted with the fibroblasts expression empty vector. In contrast, the fibroblasts over-expressing Cbfa l gene failed to stimulate bone regeneration, compared with that transplanted with the fibroblasts expression empty vector.