人骨形成蛋白9基因修饰的兔骨髓间充质干细胞异位成骨实验研究  

Ectopic Bone Formation of Bone Marrow Mesenchymal Stem Cells with Gene Transfer of Human Bone Morphogenetic Protein-9 Gene in Rabbit

作　　者：江标[1] 李明[1] 曹豫江[1] 

机构地区：[1]重庆医科大学附属儿童医院骨科,重庆400014

出　　处：《四川大学学报（医学版）》2008年第5期723-727,共5页Journal of Sichuan University(Medical Sciences)

摘　　要：目的探讨人骨形成蛋白9(human bone morphogenetic 9,hBMP-9)基因治疗与骨组织工程技术相结合的可行性。方法采用阳离子脂质体介导hBMP-9基因转染兔骨髓间充质干细胞(mesenchymal stem cells,MSCs),荧光显微镜观察及流式细胞学检测,碱性磷酸酶(ALP)活性定量测定及Von Kossa’s钙结节染色;MSCs与聚乳酸-羟基乙酸(polylactide-co-glycolide,PLGA)支架共培养,荧光显微镜及扫描电镜观察MSCs在PLGA上的黏附、生长情况;将转染与未转染hBMP-9基因的MSCs分别与PLGA支架复合培养,构建组织工程化骨并回植兔肌肉内,组织植入4、8周后进行HE染色观察。结果流式细胞仪测定质粒转染率为34.15%;转染组MSCs的ALP活性较未转染组明显增高(P<0.01),Von Kossa’s钙结节染色比较转染细胞形成的钙结节明显大于未转染组;荧光显微镜及扫描电镜观察见MSCs在PLGA支架上的黏附、生长良好;异位回植术后4、8周组织HE染色见肌肉内有新生骨基质分泌,成骨面积定量分析转染组与对照组相比差异有统计学意义(P<0.05)。结论阳离子脂质体介导的质粒能稳定转染MSCs,BMP-9能刺激MSCs的ALP活性增强,诱导钙结节形成;BMP-9基因转染的MSCs作为一种新型的种子细胞,其与PLGA支架复合用于骨缺损的修复具有较强的可行性。Objective To investigate the method combining hBMP-9 gene therapy with tissue-engineering techniques to improve osteogenesis in an ectopic bone formation model in rabbits. Methods Rabbit marrow MSCs were transferred with BMP-9 gene by cationic liposome, and then were subjected to a series tests including fluorescent microscope, Flow cytometer （FCM）, ALP activity quantitative assay and Von Kossa＇s calcium nodus staninig; MSCs transfected with BMP-9 gene successfully were seeded onto scaffolds of polylactide-co-glycolide （PLGA）. Cell-matrix interactions were observed with fluorescent microscopy and scanning electronic microscopy. The tissue-engineered bones with MSCs seeded on PLGA were further subcutaneously implanted into rabbits. The implants were evaluated with histological staining at 4 and 8 weeks after surgery. Results The gene transfer efficiency of MSCs transfected with BMP-9 gene was 34.15%, which was measured by FCM. The ALP activity of MSCs with BMP-9 gene transfer was higher than that of non-transfered cells （P〈0. 01）. The calcium nodus formation of MSCs was enhanced by the gene modification of BMP-9 gene. MSCs seeded onto PLGA showed high level of cell proliferation, and efficient synthesis of cell matrix was observed with scanning electronic microscopy. In the eetopie bone formation model, new bone area was also significantly improved by BMP-9 gene modified MSCs seeded on PLGA （P〈0. 05）. Conclusion hBMP-9 gene modified MSCs could enhance ectopic new bone formation in rabbits. These results indicated that the strategy combining BMP-9 gene modified MSCs with 7PLGA might be suitable for bone tissue engineering applications.

关 键 词：骨髓间充质干细胞 BMP-9 PLGA 组织工程 

分 类 号：R68[医药卫生—骨科学]