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

发布时间：2018-03-19 05:00

本文选题：腺病毒　切入点：骨形态发生蛋白　出处：《中国骨质疏松杂志》2014年08期 　论文类型：期刊论文

【摘要】：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。
[Abstract]:Objective to transfect human bone morphogenetic protein-2 and EGFP gene into rabbit bone marrow stromal stem cells (BMSCs) by adenovirus vector, and to construct tissue engineered bone with DBM (decalcified bone) scaffold in vitro. Methods Rabbits bone marrow stromal cells were isolated and cultured. After transfection, the expression of exogenous genes was detected by fluorescence microscopy and Western blotting. The method provided by Urist was used to prepare decalcified DBM. The adhesion rate of BMSCs and DBM co-cultured was measured by cell metrology. Then the transfected cells were inoculated into DBM scaffold, and fluorescence microscope was used to observe whether the cells adhered to the scaffold material successfully. Results BMSCs cell phenotypic identification showed that the positive expression of CD44 was negative, and the expression of BMP-2 was increased by Western blotting after transfection. The average adhesion rate of bone marrow mesenchymal stem cells to decalcified bone matrix was 72.74 卤1.99%. Conclusion BMSCs can be transfected with Ad-BMP-2 / EGFP successfully. The transfected cells grow well after implantation on DBM scaffold, and the tissue engineering bone is successfully constructed.
【作者单位】：桂林医学院;桂林医学院附属医院;
【基金】：国家自然科学基金资助项目(31160199)
【分类号】：R318

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