人骨形成蛋白7基因修饰的兔骨髓基质干细胞异位成骨实验.doc

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人骨形成蛋白 7 基因修饰的兔骨髓基质干细胞异位成骨实验作者：闫露，罗二平，鱼兵，栗艳，沈敏，范清宇【摘要】目的］［通过人骨形成蛋白 7(human bone morphogenetic protein 7，BMP7)基因修饰的兔骨髓基质干细胞(mesenchymal stem cells,MSCs) 与聚乳酸 - 羟基乙酸 (polycleclide co plycoclide,PLGA)支架复合，进行自体皮下异位成骨实验，探索 BMP-7 基因治疗与组织工程技术相结合的可行性。［方法］PLGA 与腺病毒 Ad-BMP-7 感染后的兔 MSCs 共培养，电镜观察了解细胞在 PLGA 上黏附、生长和合成分泌骨基质成分的情况；将转染基因的细胞和未转染基因的细胞分别与 PLGA 支架复合，构建组织工程化骨并植入体内，通过组织观察新骨形成情况。［结果］制备得到的 PLGA 为疏松多孔的网格样结构，具有一定的韧性和强度。将腺病毒感染的骨髓基质干细胞接种于修剪成一定形状的 PLGA 上，电镜观察可见细胞贴附于材料表面和经孔隙内并增殖。异位成骨实验证实空白对照 PLGA 孔隙内无新骨形成，对照细胞组内有部分新生骨形成， Ad 而 BMP 7 实验组形成的新生骨组织面积更大(P<0.05)。论］［结应用 hBMP 7 基因修饰的 MSCs 作为骨组织工程的种子细胞，可望取得更强的成骨能力。【关键词】基因治疗；骨形成蛋白；组织工程 1 Abstract： Objective］hBMP ［ 7 gene therapy combined with tissue engineering techniques was explored to improve osteogenesis in an ectopic bone formation model in rabbits. ［Method］PLGA(with diameter of 150～300 mm and porosity of 90%) was prepared and autologous bone marrow stromal cells(bMSCs) infected by adenovirus carring hBMP 7 gene(Ad BMP 7) were seeded onto it.Scanning electronic microscoping were used to observe cell matrix interaction.The cells infected and non infected were combined with PLGA at a concentration of 5× 106cells/ml to construct tissue engineered bones respectively.They were further implanted into 8 rabbits subcutaneously using PLGA as blank control(8 implants per group). Six weeks after surgery, the implants were evaluated with histological staining and computerized analysis of new bone formation.［Result］PLGA prepared was a grid like porous structure, with definite ductility and strength,and could be trimmed into different models. Adenovirus infected MSCs seeded onto PLGA showed high level of proliferation, and mass synthesis of cell matrix was observed with electronic microscoping.In the ectopic bone formation model, PLGA alone could not induce new bone formation. The new bone area formed 2 in the non infected MSCs group was 28.6±6.1%,while Ad BMP 7 group was 57.5±5.7%,which was higher than that of the former significantly(P<0.05).［Conclusion］hBMP 7 gene modified MSCs could enhance ectopic new bone formation in rabbits. These results indicated that the infection of MSCs with hBMP 7 adenovirus might be suitable for bone tissue engineering applications. Key words：gene therapy； bone morphogenetic protein； tissue engineering 由支架材料、种子细胞和生长因子复合构建的组织工程骨为临床治疗顽固性的骨折不愈合和大型骨缺损带来了希望。然而，如何实现生长因子在特定的解剖部位持续稳定的释放，这个问题一直未得到有效解决。骨髓基质干细胞(mesenchymal stem cells, MSCs)具有明确的成骨潜能，是骨愈合过程中骨化的重要细胞。它具有来源丰富，采集方便，容易在体外培养、诱导、扩增的特点，是组织工程化骨理想的种子细胞。该细胞在体外培养具有较强的传代增殖能力，外源目的基因易于导入，因此也是骨缺损基因治疗较为理想的靶细胞［1］。本实验利用腺病毒介导 hBMP 7 基因转染 MSCs 并与具有良好生物相容性和生物降解特性的聚合物 PLGA 复合，构建组织工程化骨，并进一步进行兔自体皮下异位成骨实验，以探讨 BMP 7 基因治疗与组织工 3

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