人骨形成蛋白4基因修饰的组织工程化骨的实验研究

The study of hBMP-4 gene modified tissue-engineered bone

目的 探讨基因治疗与组织工程方法相结合促进成骨的效果。方法 应用亚克隆法构建真核表达载体 pEGFP hBMP 4。贴壁法培养兔骨髓基质干细胞 (bonemarrowstromalcells,bMSCs) ,体外转染pEGFP hBMP 4 ,pEGFP基因并留置空白对照 ,以天然型无机骨为支架材料 ,分别构建组织工程化骨 ,植入裸鼠皮下 (每组 6例 ) ,4周取材 ,进行组织学及新骨成骨面积定量分析。结果成功构建了序列完全正确的 pEGFP hBMP 4表达载体。bMSCs可以在天然型无机骨支架网孔内表面贴壁生长。裸鼠体内实验表明术后 4周转染pEGFP hBMP 4基因组支架材料中已大部由新骨充满 ,新骨面积大于对照组 (P <0 0 5 )。结论 应用hBMP 4基因转染的bMSCs作为种子细胞构建组织工程化骨 。

Objective Bone marrow stromal cells (bMSCs) of rabbits transferred with mammalian hBMP-4 expression plasmid were used to construct tissue-engineered bone. Gene therapy combined with tissue-engineering technique was explored to further improve osteogenesis. Methods pEGFP-hBMP-4 plasmid was constructed by subcloning technique. bMSCs were then transferred with either pEGFP-hBMP-4, pEGFP plasmid by lipofectamine or left uninfected in vitro. The cells from the 3 groups were combined with natural non-organic bone (NNB) to construct tissue-engineered bones, which were subcutaneously implanted into nude mice (6 implants per group) for 4 weeks. Specimens were evaluated through histological and computerized new bone formation analysis. Results pEGFP-hBMP-4 plasmid was successfully constructed. bMSCs could attach and proliferate on the surface of NNB. In vivo experiment showed that new bone formation in pEGFP-hBMP-4 group was higher than those of the control groups. Conclusions Tissue-engineered bone using hBMP-4 gene modified bMSCs might be an ideal alternative for the repair of bone.