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

我们进行了以下实验。骨再生过程中间充质干细胞的表征我们首先在生长小鼠的膈肌上做一个直径为2.2 mm的圆孔。为了研究骨修复过程中间充质细胞的增殖活性，我们探索了BrdU的掺入。这些研究表明，在我们的实验系统中，含有BrdU的间充质细胞在损伤后3天增殖增加。骨再生过程中基因表达的变化利用上述实验模型，通过Northern blot分析骨相关基因表达模式的变化。骨损伤后第4天ALP mRNA升高，第5天骨钙素mRNA升高。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，我们的实验模型在损伤后第4天和第5天出现了间充质干细胞。我们正在利用CDNA芯片探索与间充质干细胞相关的基因。为了开发新的治疗方法，我们将皮肤成纤维细胞移植到带载体（PGS）的生长小鼠颅骨骨缺损（直径2.2 mm）中。过表达BMP-2 CDNA的成纤维细胞移植比用表达空载体的成纤维细胞移植更能促进骨再生。相比之下，与移植成纤维细胞表达空载体相比，过度表达Cbfa - 1基因的成纤维细胞未能刺激骨再生。

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.