人骨髓间充质干细胞向成骨细胞和成软骨细胞分化的能力

背景:利用骨髓间质干细胞(mesenchymalstemcells,MSCs)的多向分化性,选择合适的生物材料和适当的生长因子联合应用,可达到修复组织缺损的目的,MSCs细胞具有取材方便,对身体健康损害小,来源于自体干细胞诱导构建的组织不存在MHC限制等优点,所以日益受到人们的重视。目的:探讨人骨髓间充质干细胞向成骨和成软骨细胞诱导分化的能力。设计:单一样本研究。单位:苏州大学附属儿童医院骨科,苏州大学生命科学院生物技术研究所。材料:RPMI1640完全培养基,地塞米松,β-甘油磷酸钠,抗坏血酸,鼠抗人一抗,羊抗鼠二抗,链球菌亲和素(三抗),DAB(1:50),100mL/L胎牛血清,维生素C,转化生长因子-β1(TGF-β1)等。方法:选用不同代次的MSCs细胞,分别使用成骨和成软骨条件培养基培养,观察细胞的形态学变化,同时采用细胞化学和免疫组化的方法检测碱性磷酸酶,钙盐沉积,糖胺多糖分泌和I,Ⅱ胶原的表达。主要观察指标:骨髓基质干细胞向成骨细胞分化;骨髓基质干细胞向成软骨细胞分化。结果:MSCs细胞在地塞米松、维生素C和β-甘油磷酸钠的作用下,15d后可见细胞变为多边形,ALP和I型胶原染色阳性,形成钙结节,表现成骨细胞分化特点;而在维生素C和TGF-β1的作用下,7d可见细胞多为圆形,糖胺多糖和II型胶原染色阳性,表现成软骨细胞分化特点。

Differentiationpotential of human mesenchymal stem cells into osteoblasts and chondroblasts

BACKGROUND: The repair of the tissue defect can be achieved by utilizing the multiple-direction differentiation of mesenchymal stem cells (MSCs), which can be applied with the combination of appropriate biomaterials and appropriate growth factors. MSC has some advantages, like convenient source of materials, small harm to human health, and originating from the tissues induced and constructed by autologous stem cells without limitation of MHC. Hence, it gradually receives recognitions.OBJECTIVE: To explore the potential of human marrow mesenchymal stem cells(hMSCs) differentiating into osteoblasts and chondroblasts.DESIGN: A single sample study.SETTING: Department of Orthopaedics, the Affiliated Children' s Hospital of Soochow University and Institute of Life Sciences and Biotechnology,Soochow University.MATERIALS: RPMI1640 complete medium, dexamethasone,β-glycerophosphate, ascorbate, mouse-anti-human primary antibody,goat-anti-mouse secondary antibody, streptavidin(tertiary antibody), DAB (1: 50), 100 mL/L of fetal calf serum, vitamin C, transforming growth factor β1 (TGF-β1), etc.METHODS: Different generations of MSCs were selected and cultured separately in culture medium containing osteoblasts and chondroblasts for the observation of the cellular morphological changes. Alkaline phosphatase (ALP) activity, calcium deposition secretion of glycosaminoglycan and expression of type Ⅰ and Ⅱ were determined by cytochemistrical and immunohistochemical methods.MAIN OUTCOME MEASURES: Differentiation of MSCs into osteoblasts and differentiation of MSCs into chondroblasts.RESULTS: MSCs became polygon cells after 15 days under the inductions of dexamethasone, vitamin C and β-glycerophosphate medium. ALP and type Ⅰ collagens staining were positive. Calcified nodule could be seen,which represented an osteogenic potential. MSCs also differentiated into chondrocytes after exposure to vitamin C and TGF-β1 for 7 days, the cells became round, glycosaminoglycan and type Ⅱ collagen staining were positive, which represented a chondrogenic potential. However, the expressions of cellular differentiation had a weakening tendency with cellular proliferation.CONCLUSION: The successful induction of hMSCs derived from bone marrow differentiating into osteoblasts and chondrocytes under certain culture condition provides a foundation for further researches.