腺病毒介导 BMP－2和 EGFP 基因转染兔骨髓基质干细胞及种植脱钙骨的体外观察

摘要

Objective To construct the tissue engineering bone in vitro by implanting the rabit bone marrow stromal cells ( rBMSCs) , which were infected by a recombinant adenoviral vector carrying human BMP-2 and EGFP gene ( Ad-hBMP-2/EGFP) , into DBM.Methods The rBMSCs were isolated and cultured in vitro.The cell membrane markers were detected using flow cytometry.After transfection, the optimal multiplicity of infection ( MOI) of BMSCs was observed using fluorescence microscopy.The expression of exogenous gene in the cells was detected using Western blotting.The allogenic DBM was prepared according to the method described by Urist.After co-cultured with DBM in vitro, the adhesion rate of BMSCs on DBM was tested using cytometry.Then the transfected BMSCs were seeded on DBM.The fluorescence microscopy was used to observe whether the BMSCs successfully adhered to the DBM.And the attachment and growth of the cells on the scaffold were examined using SEM.Results The phenotype identification of BMSCs showed that the expression of D44 was positive, while the expression of CD45 was negative.After transfection, the expression of hBMP-2 increased, which was confirmed by Western blotting.The average adhesive rate of BMSCs on DBM was 72 .74%±1.99%.SEM examination revealed extensive cellular attachment and growth on the DBM.Conclusion The culture and identification of rBMSCs are successful.The rBMSCs are successfully transfected with Ad-EGFP-BMP2, with high efficiency expression of objective gene.The growth of the transfected cells seeded on DBM is good, indicating the successful construction of tissue engineering bone.%目的：用腺病毒载体介导人骨形态发生蛋白－2及EGFP基因转染兔骨髓基质干细胞（ rBMSC），种植DBM （脱钙骨）支架体外构建组织工程骨。方法兔髓基质干细胞（ rBMSC）的分离、培养；流式细胞仪检测细胞表面标记；转染后，荧光显微镜观察细胞最适感染复数（MOI）及蛋白印迹检测外源基因的表达情况；采用Urist提供的方法制备脱钙骨（DBM），用细胞计量法测定BMSCs与DBM复合培养的黏附率。然后将转染后细胞接种到DBM支架上，用荧光显微镜观察细胞是否成功粘附于支架材料上，扫描电镜观察细胞贴附、生长状况。结果 BMSCs细胞表型鉴定：CD44表达阳性，CD45表达阴性；转染后，蛋白印迹试验检测到BMP－2表达增高；骨髓间充质干细胞与脱钙骨基质的平均粘附率为（72．74±1．99）％；扫描电镜见转染细胞生长良好，伸出丝状突起，相互连接。结论成功培养及鉴定兔BMSCs，Ad－BMP－2／EGFP可高效转染兔BMSCs，转染后的细胞种植于DBM支架材料后生长状况良好，组织工程骨构建成功。