骨形态发生蛋白-2基因转染兔骨髓基质干细胞及复合纳米羟基磷灰石体外构建组织工程骨的研究

Rabbit bone marrow stromal cells transfeeted by adenovirus mediated bone morphogenetic protein-2 gene plus nano-hydroxyapatite to reconstruct tissue engineered bone in vitro

目的探讨腺病毒介导的人骨形态发生蛋白-2表达载体（Ad—BMP-2）转染兔骨髓基质干细胞（rBMSCs）后复合到纳米羟基磷灰石（Nano-HA）体外构建仿生人工骨的可行性。方法采用GatewayTM技术构建腺病毒载体，溶胶-絮凝法制备Nano-HA，用Ad—BMP-2转染体外培养的rBMSCs，免疫组化、RT—PCR和蛋白印迹方法检测转染后细胞的BMP-2表达。将转染48h的细胞均匀接种到Nano—HA支架上，第3、5天取细胞载体复合物扫描电镜观察细胞贴附、生长状况，消化收集贴附支架的细胞，蛋白印迹检测贴附支架细胞BMP-2的表达。结果转染后BMP-2基因在mRNA水平和蛋白水平均有高表达；扫描电镜见转染细胞在支架材料分布均匀，黏附良好，转染后及复合后Western Blot检测BMP-2表达较高。结论利用转基因技术，用含目的基因的腺病毒载体转染靶细胞复合到Nano—HA，细胞载体复合物稳定长效地分泌生长因子，体外成功地构建了仿生人工骨。

Objective To investigate the effects of adenovirus-mediated human bone morphogenetic protein-2 expression vector（Ad-BMP-2） transfected with rabbit bone marrow stromal cells（rBMSCs） plus nano-hydroxyapatite（Nano-HA） in reconstruction of biomimetic artificial bone in vitro. Methods The adenovirus vector was constructed by GatewayTM technology. Nano-HA was prepared by Sol-Flocculation. rBMSCs transfected by Ad-BMP-2 were cultured in vitro. Immunohistochemistry, RT-PCR and Western Blot were used to detect the BMP-2 expression in the transfeeted cells. After culture for 48 h, the transfected cells were seeded onto the Nano-HA scaffold. Scanning electron microscopy was used to observe the growth and attachment of cells on the composite scaffold on the third and fifth days. The attached cells were also gathered on the third and fifth days for detection of the BMP-2 expressions by Western Blot. Results After transfection, bBMP-2 gene showed high expressions in mRNA and protein levels. Scanning electron microscopy revealed even distribution and fine adherence of the cells in the composite scaffold. Western Blot showed high expression of BMP-2 in the transfected and compound cells. Conclusion The Ad-BMP-2 transfected with rBMSCs plus Nano-HA can successfully reconstruct the in vitro biomimetic artificial bone which can secrete growth factors stably.