重组PcDNA3.1-hBMP-2转染骨髓基质干细胞及复合异种骨支架体外构建组织工程骨

目的：构建人骨形态发生蛋白-2(human bone morphogenetic protein,，hBMP-2)真核表达载体PcDNA3．1-hBMP-2，转染兔骨髓基质干细胞(bane marrow stromal cells，BMSCs)，种植去抗原牛松质骨(bovine cancellous bone，BCB)支架体外构建组织工程骨。方法：蛋白印迹法检测转染后细胞BMP-2的表达，碱性磷酸酶(ALPase)活性检测分析基因转染对细胞分化的影响。然后将转染后细胞接种到BCB支架上，扫描电镜观察细胞贴附、生长状况。结果：转染后，BMSCs表达BMP-2，ALP活性明显增高。扫描电镜见转染细胞分布均匀，伸展良好。结论：在脂质体介导下，BMP-2基因可导入细胞且稳定表达基因产物促进自身增殖分化，转染后细胞在支架材料上贴附生长良好，为进一步应用携带BMP-2基因的人工骨修复骨缺损奠定了实验基础。

Construction of tissue engineering bone with hBMP-2 gene transfected bone marrow stromal cells and bovine cancellous bone scaffold in vitro

Objective:To construct rabbit bone marrow stromal cells (BMSCs) transfecting by a recombinant plasmid carrying the human bone morphogenetic protein-2 gene (PcDNA3.1-hBMP-2) seeded into bovine cancellous bone (BCB) scaffolds to construct tissue engineering bone in vitro.Methods:The expression of hBMP-2 in these cells after transfection was determined by Western blot analysis.The changes of cellular differentiation were observed by ALPase activity analysis.BMP-2 transduced cells were then seeded into BCB scaffolds.The attachment and growth of the cells on the scaffold were examined using scanning electron microscope (SEM).Results:The expression of hBMP-2 was confirmed and ALPase activity obviously increased in the cells after transfection.SEM examination revealed extensive cellular attachment and growth on the BCB block.Conclusion:With the help of lipofectamine,the transfection of PcDNA3.1-hBMP-2 to BMSCs is carried out successfully.The cells after transfection grow well on a BCB scaffold.Tissue engineering bone used to regional gene therapy is constructed successfully.