BMP-2基因转染的人骨髓基质干细胞复合PLA/PCL支架体外构建组织工程骨

目的　用人骨形态发生蛋白 2腺病毒表达载体 (Ad -BMP - 2 )转染的人骨髓基质干细胞 (hBMSC) ,复合PLA/PCL(聚乳酸 /聚己内酯 )生物降解支架体外构建组织工程骨。方法　用Ad -BMP - 2转染体外培养的成人BMSC ,免疫组化、原位杂交染色和蛋白印迹方法检测细胞BMP - 2的表达 ,并通过流式细胞仪和ALP活性检测分析其对细胞增殖、分化的影响。然后将转染后细胞接种到PLA/PCL支架上 ,扫描电镜观察细胞贴附、生长状况。结果　转染后 ,hBMP - 2基因在mRNA水平和蛋白水平均有表达 ;S期细胞比例和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　Ad-BMP - 2可高效转染hBMSC ,且促进细胞增殖及成骨转化。转染后细胞在PLA/PCL上生长良好 ,BMP - 2基因治疗的组织工程骨构建成功

Construction of Tissue Engineering Bone with BMP-2 Gene Transfected Human Bone Marrow Stromal Cells and PLA/PCL Biodegradable Scaffold in Vitro

Objective Human bone marrow stromal cells (hBMSC) infected by a recombinant adenoviral vector carrying the human BMP-2 gene (Ad-BMP-2) were seeded into PLA/PCL biodegradable scaffolds to construct tissue engineering bone in vitro.Methods Ad-BMP-2 infected hBMSC cultured in vitro and the expression of hBMP-2 in these cells after infection was determined by in situ hybridization and immunohistochemical analysis.BMP-2 productions were confirmed by western blot analysis of the supernatant collected from the cells.The changes of cellular proliferation and differentiation in the cells were observed by flowcytometry and ALP activity analysis.BMP-2 transduced cells were then seeded into PLA/PCL biodegradable scaffolds.The attachment and growth of the cells on the scaffold were examined using SEM.Results The expression of hBMP-2 was confirmed in mRNA and protein levels in the cells after infection and the presence of BMP-2 was detected in the supernatant of the cells.In addition,cellular proportion in S period and ALP activity obviously increased in the cells.SEM examination revealed extensive cellular attachment and growth on the PLA/PCL composite in 1 day.Conclusion Ad-BMP-2 could infect hBMSC with high efficiency and promote cellular proliferation and osteoblast conversion.The cells after infection grew well on a PLA/PCL scaffold.Tissue engineering bone used to regional gene therapy is constructed successfully.