人重组骨形态发生蛋白9基因修饰的兔骨髓间充质干细胞异位成骨实验研究

目的 探讨人重组骨形态发生蛋白9(human recombinant bone morphogenetic 9,rh-BMP-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rhBMP-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Vbn 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上的粘附、生长情况.将转染与未转染rhBMP-9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周后进行HE染色观察.结果 菜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支架上的粘附、生长良好.异位回植术后4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,rhBMP-9能刺激MsCs的ALP活性增强,诱导钙结节形成,rhBMP-9基因转染的MSCs作为一种新型的种子细胞,其与PUGA支架复合用于骨缺损的修复具有较强的可行性.

Ectopic bone formation with rhBMP-9 transfected bone marrow mesenchyml stem cells infusion in rabbit

Objective Human bone morphogenetic protein-9 (hBMP-9) gene therapy combined with tissue-engineering techniques was employed to improve osteogenesis in rabbit model. Methods Rabbit marrow mesenehymal stem cells (MSCs) were transfected with rhBMP-9 gene. Fluorescent mi-croscope,flow cytometer (FCM),ALP activity quantitative assay and Von Kossa's calcium nidus stai-ning were carried out. PLGA was prepared and MSCs were transfected with BMP-9 gene cationic lipo-somes. Scanning electronic microscopy as used to study cell matrix interaction. The infected and the non-infected cells were combined with PLGA to construct bone tissues respectively. They were further implanted into rabbits subcutaneously. Four weeks after surgery, the implants were evaluated with his-tological staining. Results The transfection efficiency of MSCs was 34.15%. The ALP activity of in-fected MSCs was higher than that of non-infected cells (P<0.01 ). The calcium niduses of BMP-9 transfected MSCs group were significantly larger than those of the non-infected group. MSCs seeded onto PLGA proliferated rapidly. Synthesis of celt matrix was observed with scanning electronic micros-copy. The new bone formation activity of the non-infected MSCs group was lower than the BMP-9 in-fected group (P<0.05). Conclusions hBMP-9 infected MSCs could enhance ectopic bone formation in rabbits. This is potentially a suitable approach for bone tissue engineering.