骨髓基质干细胞体内成骨分化研究

发布时间：2018-05-18 19:25

本文选题：骨髓基质干细胞 + 细胞分化　；参考：《中国协和医科大学》2008年博士论文

【摘要】： 目的:探讨自体骨髓基质干细胞移植在股骨头缺损坏死部位,移植的骨髓细胞能否成活,能否停留在骨坏死、缺损部位生长增殖,能否分化为成骨细胞,及能否促进成骨、修复股骨头缺损坏死。方法:试验分三部分。①抽取双侧狗髂骨骨髓。骨髓细胞行贴壁细胞分离法分离骨髓基质干细胞(BMSCs),进行体外培养扩增,试验用第二代细胞。移植前,用5-溴脱氧尿嘧啶核苷(BrdU)标记BMSCs。在狗双侧股骨头后外侧软骨面近头颈交界处,用直径8mm的环钻向股骨头中心方向钻孔10mm深度,造成股骨头直径8mm、深度10mm的骨缺损模型。环钻取出的骨质经微波灭活后,与标记的BMSCs复合,植入一侧自体股骨头缺损处,另一侧单纯植入微波灭活骨作为对照,术后5周取材。②抽取双侧髂骨骨髓,行密度梯度离心法分离BMSCs,用荧光染料羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)标记BMSCs,用明胶海绵吸附分离出的BMSCs,植入一侧自体股骨头缺损处,另一侧单纯植入明胶海绵作为对照,术后3周取材。③将体外培养扩增的BMSCs,与脱抗原小牛松质骨复合植入一侧自体股骨头缺损处,另一侧单纯植入脱抗原小牛松质骨作为对照,术后12周取材。利用激光共聚焦显微镜和免疫组织化学法分别观察CFSE和Brdu的标记和体内定位情况。行X射线片,大体组织观察,组织化学染色,图像分析,及通过免疫组织化学方法和荧光免疫组织化学方法检测成骨细胞特异性分化标记核心结合因子a1(Cbfa1)、骨钙素、骨桥素和Ⅰ型胶原,以了解BMSCs体内分化状况和成骨效果。结果:大体组织学观察显示,BMSCs移植侧骨缺损成骨程度明显强于对照侧。显微组织学研究的结果进一步显示,BMSCs移植侧骨缺损区内的成骨量、骨基质矿化的程度、骨成熟度明显高于对照侧,含有更加丰富的梭形间充质细胞和成骨细胞,成骨细胞分化特异性标记Cbfa1、骨钙素、骨桥素、Ⅰ型胶原表达也明显强于对照侧。股骨头缺损内发现大量BrdU阳性细胞,新骨形成越活跃的地方,BrdU阳性细胞数目越多,很多成骨细胞BrdU阳性,新骨形成区的很多血管壁也分布着BrdU阳性细胞。荧光显微镜下观察,见BMSCs移植侧带有CFSE绿色荧光的细胞定位于骨缺损内,与抗RunX2、Osteocalcin、Osteopontin一抗反应的二抗(带罗丹明标记)在绿色荧光集中的地方也集中发出红色荧光。结论:移植的骨髓细胞在股骨头内能成活,并能停留在骨缺损部位生长增殖。移植的BMSCs能分化成为多种细胞,包括成骨细胞、软骨细胞及血管壁细胞等。BMSCs能分化成成骨细胞参与成骨,也能参与血管形成。移植的BMSCs能显著促进骨坏死和骨缺损的修复。
[Abstract]:Objective: to investigate whether autologous bone marrow stromal cells can survive, remain in osteonecrosis, grow and proliferate, differentiate into osteoblasts and promote osteogenesis after transplantation of bone marrow stromal cells in the necrotic site of femoral head. Repair of femoral head defect and necrosis. Methods: the bone marrow of bilateral iliac bone was extracted in three parts. Bone marrow mesenchymal stem cells (BMSCs) were separated by adherent cells and cultured in vitro. The second generation cells were used in the experiment. BMSCs were labeled with 5-bromodeoxyuridine (BrdU) before transplantation. At the point of bilateral femoral head posterolateral cartilage near the junction of head and neck, the 10mm depth of diameter 8mm was drilled to the center of femoral head, and the bone defect model with diameter of 8mm and depth of 10mm was made. After the bone was inactivated by microwave, the bone was combined with labeled BMSCs and implanted into the defect of one side of the femoral head, and the other side was simply implanted into the microwave inactivated bone as a control. The bone marrow of bilateral iliac bone was extracted from the bone marrow of bilateral ilium 5 weeks after operation. BMSCs were separated by density gradient centrifugation. BMSCs were labeled with fluorescent dye carboxyl fluorescein diacetate succinimide (CFSE). BMSCs were adsorbed by gelatin sponge and implanted into the defect of one side of femoral head. The other side was simply implanted with gelatin sponge as control. Three weeks after operation, the expanded BMSCs were implanted into the defect of one side of the femoral head and the other side was used as the control. 12 weeks after the operation, the samples were taken from the cultured calf cancellous bone and the deantigen calf cancellous bone were implanted into the defect of one side of the femoral head. The labeling and in vivo localization of CFSE and Brdu were observed by laser confocal microscopy and immunohistochemistry respectively. X-ray, gross tissue observation, histochemical staining, image analysis, and detection of osteoblast specific differentiation marker core binding factor A1Cbfa1, osteocalcin, and osteocalcin were performed by immunohistochemistry and fluorescence immunohistochemistry. Osteopontin and type I collagen were used to understand the differentiation and osteogenic effect of BMSCs in vivo. Results: gross histological observation showed that the osteogenic degree of bone defect of BMSCs was significantly stronger than that of control. The results of microhistology further showed that the amount of bone formation, the degree of mineralization of bone matrix and the maturity of bone in the bone defect area of BMSCs were significantly higher than those in the control side, and contained more fusiform mesenchymal cells and osteoblasts. The expression of Cbfa1, osteocalcin, osteopontin and type I collagen in osteoblasts was significantly higher than that in the control group. A large number of BrdU positive cells were found in the defect of femoral head. The more active the new bone formation was, the more BrdU positive cells were, and many osteoblasts were positive for BrdU. BrdU positive cells were also distributed in many vascular walls of the new bone formation area. Under the fluorescence microscope, it was found that the cells with green fluorescence of CFSE in the BMSCs graft were located in the bone defect, and the second antibody (labeled with Rhodamine), which reacted with the first antibody against RunX2Osteocalcinn Osteopontin, also emitted red fluorescence in the place where the green fluorescence was concentrated. Conclusion: the transplanted bone marrow cells can survive in the femoral head and remain at the site of bone defect. BMSCs can differentiate into a variety of cells, including osteoblasts, chondrocytes and vascular parietal cells. BMSCs can differentiate into osteoblasts and participate in angiogenesis. Graft BMSCs can significantly promote bone necrosis and bone defect repair.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329;R68

【引证文献】