【作者】 张勇；

【导师】 范清宇；

【作者基本信息】 第四军医大学 ， 外科学， 2006， 博士

【摘要】 目的 以AdEasy腺病毒表达系统制备携带Cbfa1基因的高滴度腺病毒，感染小鼠成肌细胞C2C12，利用外源性基因编码的生长因子诱导其表达成骨细胞表型。再将感染的C2C12与高分子生物材料chronOS支架相复合，在体外构建组织工程化骨，移植修复小鼠股骨骨缺损，观察该方法的可行性和有效性，以期找到一种较好的修复骨缺损的方法。 方法 (1)将小鼠Cbfa1基因cDNA克隆到穿梭载体形成转移质粒pAdTrack-CMV-Cbfa1，经PmeⅠ酶切线性化后与骨架质粒感受态细胞AdEasier-1 Cells经电穿孔法同源重组得到腺病毒质粒pAdCbfa1并酶切鉴定，PacⅠ酶切线性化后感染293细胞包装后获得复制缺陷型重组腺病毒AdCbfa1。(2)以复制缺陷重组腺病毒介导小鼠Cbfa1基因感染C2C12后，以MTT法及流式细胞仪观察感染对细胞增殖活力及细胞周期的影响；用RT-PCR法、免疫组化染色以及Westernblot证实转基因C2C12的Cbfa1蛋白和Ⅰ型胶原的表达情况；通过改良Gomori钙钴法及ALP检测试剂盒检测两组细胞的ALP活性；通过放免分析试剂盒检测两组细胞的骨钙素(osteocalcin，OCN)含量。(3)制备孔径为150～300μm，孔隙率为90％的chronOS与腺病毒感染后的C2C12更多还原

【Abstract】 Objective To clone human osteogenic sarcoma core binding factorα1 gene cDNA full length and generate high titers level recombinant adenoviruses containing hCbfal gene the replication defective recombinant adenoviruses AdEasy system was used. To induce expression of osteogenic phenotype by mCbfa1 gene the mouse C2C12cells were infected by recombinant adenovirus. Then the tissue-engineered bone was constructed by combination of infected C2C12 andchronOS and its feasibility and ability to enhance segmental bone defect healing of mouse femur was explored.Methods (1) mCbfal gene was cloned into Shuttle vector to generate pAdTrack-CMV-Cbfal, which was linearized by PmeI and co-transformed into adenoviral backbone plasmid competent cells-AdEasier-1 Cells to obtain recombinant adenoviral plasmid- pAd-Cbfa1 by homologous recombination. Recombinants were selected and confirmed by restriction endonuclease analysis. After packed in 293 cells, the recombinant adenoviruses AdCbfal were generated. (2) The proliferation and cell cyle analysis of gene-modified C2C12 were assessed by MTT method and FCM after transfected with Ad-Cbfa1. The methods of RT-PCR, immunohistochemical staining and western blot were used to test the expression of Cbfa1 protein. Gomori stain and ALP testing kit were used to measure ALP activity and immunohistochemistry testing kit were used to measure OCN contents. In addition, the collagen type I were tested with the methods of RT-PCR and immunohistochemical staining. (3)更多还原