BMP-9基因修饰的兔骨髓间充质干细胞异位成骨实验研究
摘要
创伤和手术后的骨缺损常需要植骨,故骨修复是骨科的基本问题。将编码细胞因子的基因导入骨髓间充质质干细胞(BMSCs),再与合适的载体复合后植入骨缺损部位,可持续有效地表达细胞因子,诱导BMSCs向成骨细胞分化,促进骨愈合。有研究证实,BMP-9基因转染可促进人BMSCs增殖及成骨转化。本实验在此基础上,观察BMP-9基因修饰的兔BMSCs在PLGA支架中附着、生长情况,为进一步应用此组织工程骨修复骨缺损奠定基础。将细胞与可降解类生物材料等复合物移植于缺损部位替代修复骨缺损是一种新的尝试。本实验在基因修饰骨髓间充质干细胞的基础上,将细胞与可降解生物材料PLGA复合物在体外短暂培养后移植到兔臀部肌袋内,观察其异位成骨的能力,探讨一种实用性的骨缺损组织工程修复材料。
目的:通过人骨形态发生蛋白-9(BMP-9)基因修饰兔骨髓间充质干细胞(BMSCs),接种PLGA(聚乳酸/羟基乙酸共聚物)支架体外构建组织工程骨;并将复合物移植到兔臀部肌袋内异位成骨,探索BMP-9基因治疗与组织工程技术相结合的可行性。
方法:贴壁法培养兔骨髓MSCs,体外应用阳离子脂质体介导转染pEGFP-BMP-9基因:荧光显微镜观察及流式细胞学检测观察转染效率;MSCs碱性磷酸酶活性半定量测定及Von Kossa’s钙结节染色观察转染前后细胞功能变化;将转染及未转染基因的MSCs与PLGA支架共培养,体外构建组织工程骨,荧光显微镜及扫描电镜观察MSCs在PLGA支架上的黏附、生长状况。短暂体外培养后手术将MSCs-PLGA复合物分组移植到兔臀部肌袋内;于不同时间点(2W、4W、8W)行X线摄片和组织切片行HE染色观察骨基质的生成情况,及进行新骨成骨面积分析,并于(4W、8W)进行组织标本ALP测定。
结果:流式细胞学测定阳离子脂质体介导的质粒转染率为34.15%;转染组MSCs的ALP活性较未转染组明显增高(p<0.05),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。异位成骨4W X线摄片见实验组有骨组织成像显示,8W后显影增大,对照组4W时未见显影,8W时可见模糊显影出现;4W、8W组织切片HE染色见: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,而BMP-9实验组形成的新生骨组织面积更大(P<0.05)。4W、8W组织ALP检测显示实验组表达活性明显较实验对照组强(P<0.05)。
结论:BMP-9基因转染BMSCs,可促进细胞向成骨细胞增殖分化。转染后细胞在PLGA支架上生长良好,BMP-9基因修饰的组织工程骨构建成功。应用BMP-9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。
Objective hBMP-9 gene therapy combined with tissue-engineering techniques was explored to improve osteogenesis in an ectopic bone formation model in rabbits.
Method Autologous rabbit MSCs were cultured and transfected with pEGFP-BMP-9 gene by lipofectamine, fluorescent microscope、Flow cytometer (FCM)、ALP activity quantitative assay and Von Kossa’s calcium nodus staninig were detected; PLGA was prepared and MSCs infected by BMP-9 gene with cationic liposome were seeded onto it. Scanning electronic microscopy as used to observe cell matrix interaction. The cells infected and non-infected were combined with PLGA to construct tissue-engineered bones respectively. They were further implanted into rabbits subcutaneously. Four and eight weeks after surgery, the implants were evaluated with X-rays photographs and histological staining.
Result The transfection efficiency of MSCs infected by BMP-9 gene with cationic liposome detected by FCM was34.15%, the ALP activity of infected MSCs was higher than that of non-infected MSCs (p<0.05), the calcium nodus of MSCs formation of BMP-9 gene infected group were manifest larger than the non-infected group; PLGA prepared was a grid-like porous structure, with definite ductility and strength, and could be trimmed into different models. MSCs seeded onto PLGA showed high level of proliferation, and mass synthesis of cell matrix was observed with scanning electronic microscopy. In the ectopic bone formation model, four weeks after surgery, in the X-rays photographsthe can see bone tissue image formation of the experimental group, and the image became augmentation in eight weeks; the new bone area formed in the non-infected MSCs group was lower than the BMP-9 gene infected group (p<0.05). The ALP activity in the experiment side was obviously higher than that in the control side(p<0.05).
Conclusion BMP-9 gene modified MSCs could enhance ectopic new bone formation in rabbits. These results indicated that the MSCs transfer mediated by rhBMP-9 gene with cationic liposome combine with PLGA might be suitable for bone tissue engineering applications.
目的:通过人骨形态发生蛋白-9(BMP-9)基因修饰兔骨髓间充质干细胞(BMSCs),接种PLGA(聚乳酸/羟基乙酸共聚物)支架体外构建组织工程骨;并将复合物移植到兔臀部肌袋内异位成骨,探索BMP-9基因治疗与组织工程技术相结合的可行性。
方法:贴壁法培养兔骨髓MSCs,体外应用阳离子脂质体介导转染pEGFP-BMP-9基因:荧光显微镜观察及流式细胞学检测观察转染效率;MSCs碱性磷酸酶活性半定量测定及Von Kossa’s钙结节染色观察转染前后细胞功能变化;将转染及未转染基因的MSCs与PLGA支架共培养,体外构建组织工程骨,荧光显微镜及扫描电镜观察MSCs在PLGA支架上的黏附、生长状况。短暂体外培养后手术将MSCs-PLGA复合物分组移植到兔臀部肌袋内;于不同时间点(2W、4W、8W)行X线摄片和组织切片行HE染色观察骨基质的生成情况,及进行新骨成骨面积分析,并于(4W、8W)进行组织标本ALP测定。
结果:流式细胞学测定阳离子脂质体介导的质粒转染率为34.15%;转染组MSCs的ALP活性较未转染组明显增高(p<0.05),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。异位成骨4W X线摄片见实验组有骨组织成像显示,8W后显影增大,对照组4W时未见显影,8W时可见模糊显影出现;4W、8W组织切片HE染色见: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,而BMP-9实验组形成的新生骨组织面积更大(P<0.05)。4W、8W组织ALP检测显示实验组表达活性明显较实验对照组强(P<0.05)。
结论:BMP-9基因转染BMSCs,可促进细胞向成骨细胞增殖分化。转染后细胞在PLGA支架上生长良好,BMP-9基因修饰的组织工程骨构建成功。应用BMP-9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。
Objective hBMP-9 gene therapy combined with tissue-engineering techniques was explored to improve osteogenesis in an ectopic bone formation model in rabbits.
Method Autologous rabbit MSCs were cultured and transfected with pEGFP-BMP-9 gene by lipofectamine, fluorescent microscope、Flow cytometer (FCM)、ALP activity quantitative assay and Von Kossa’s calcium nodus staninig were detected; PLGA was prepared and MSCs infected by BMP-9 gene with cationic liposome were seeded onto it. Scanning electronic microscopy as used to observe cell matrix interaction. The cells infected and non-infected were combined with PLGA to construct tissue-engineered bones respectively. They were further implanted into rabbits subcutaneously. Four and eight weeks after surgery, the implants were evaluated with X-rays photographs and histological staining.
Result The transfection efficiency of MSCs infected by BMP-9 gene with cationic liposome detected by FCM was34.15%, the ALP activity of infected MSCs was higher than that of non-infected MSCs (p<0.05), the calcium nodus of MSCs formation of BMP-9 gene infected group were manifest larger than the non-infected group; PLGA prepared was a grid-like porous structure, with definite ductility and strength, and could be trimmed into different models. MSCs seeded onto PLGA showed high level of proliferation, and mass synthesis of cell matrix was observed with scanning electronic microscopy. In the ectopic bone formation model, four weeks after surgery, in the X-rays photographsthe can see bone tissue image formation of the experimental group, and the image became augmentation in eight weeks; the new bone area formed in the non-infected MSCs group was lower than the BMP-9 gene infected group (p<0.05). The ALP activity in the experiment side was obviously higher than that in the control side(p<0.05).
Conclusion BMP-9 gene modified MSCs could enhance ectopic new bone formation in rabbits. These results indicated that the MSCs transfer mediated by rhBMP-9 gene with cationic liposome combine with PLGA might be suitable for bone tissue engineering applications.
引文
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[10] F Parhami, SM Jackson, Y Tintut Y, et al, Atherogenic Diet and Minimally Oxidized Low Density Lipoprotein Inhibit Osteogenic and Promote Adipogenic Differentiation of Marrow Stromal Cells[J]. 1999; 14(12):2067-78.
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