骨髓间充质干细胞在含转化生长因子β1诱导培养基及二维培养条件下向软骨细胞的分化

背景:骨髓间充质干细胞可定向诱导为软骨细胞.目的:建立二维培养条件下骨髓间充质干细胞分化为软骨细胞的诱导体系,分析转化生长因子β 1作为诱导条件的最佳浓度,以及诱导骨髓间充质干细胞定向分化为软骨细胞的相关影响因素,观察细胞诱导后的细胞形态及表型变化.设计:随机对照动物实验.单位:贵阳医学院动物实验中心.材料:实验于2005-09/2006-11在贵阳医学院动物实验中心完成.4周龄雄性SD大鼠24只由贵阳医学院动物中心提供,体质量(98.23±7.97)g,实验过程中对动物处置符合动物伦理学标准.方法:取SD大鼠的股骨及胫骨,采用贴壁培养法分离纯化大鼠骨髓间充质干细胞,取第4代骨髓间充质干细胞行流式细胞术检测鉴定其表面抗原,分别以含1,5,10,15,20 μ g/L 的转化生长因子β 1的诱导培养基在二维培养条件下对第4代骨髓间充质干细胞诱导培养,对照组加入阴性诱导培养基.2周后采用免疫组织化学法对Ⅱ型胶原进行定性检测,采用二甲基亚甲蓝比色法定量检测诱导后细胞外基质糖胺多糖的表达.主要观察指标:各组细胞表面抗原检测、Ⅱ型胶原定性检测及细胞外基质糖胺多糖表达检测.结果:贴壁培养法可分离并纯化SD大鼠的骨髓间充质干细胞,所得第4代骨髓间充质干细胞表面抗原CD44阳性,CD34、CD45阴性.经诱导培养2周后细胞形态变为不规则,Ⅱ型胶原免疫组织化学染色显示,15,10,15,20 μg/L 转化生长因子β1组可见阳性细胞,二甲基亚甲蓝比色法检测显示各实验组细胞外基质糖胺多糖含量均明显高于对照组(P<0.01).10 μg/L 转化生长因子β 1组细胞分泌的细胞外基质糖胺多糖明显多于其它剂量组(P<0.01),并且细胞分泌细胞外基质糖胺多糖的能力与细胞密度呈正相关(r=0.822,P<0.01).结论:10 μg/L转化生长因子β 1存在的二维培养条件下,较高的细胞密度有利于骨髓间充质干细胞向软骨细胞分化.

Differentiation of bone marrow mesenchymal stem cells into chondrocytes in transforming growth factor beta-1 culture medium in two-dimensional culture in vitro

BACKGROUND: Bone marrow mesenchymal stem cells may directionally differentiate into chondrocytes.OBJECTIVE: To establish an inducing system for differentiation of bone marrow mesenchymal stem cells into chondrecytes in two-dimensional culture in vivo, to analyze tbe best concentration of transforming growth favor beta-1 (TGF-β1) to induce tbe differentiation and the correlated influence factors for the directional differentiation, and to observe the changes of cell form and phenotype. DESIGN: Randomized controlled animal study.SETTING: Animal Experimental Center, Guiyang Medical College.MATERIALS: This study was performed at Animal Experimental Center, Gulyang Medical College from September 2005 to November 2006. Twenty-four 4-week-old male SD rats weighing (98.23±7.97) g were provided by Animal Center of Guiyang Medical College. The animal experiment received informed consent from the local ethic committee.METHODS: Bone marrow mesenchymal stem cells were separated and purified from femur and tibia using attachment culture method. Surface antigen of the fourth-generated bone marrow mesenchymal stem cells was determined by using flow cytometry. Subsequendy, the fourth-generated bone marrow mesenchymal stem cells were induced with TGF-β1 culture medium (1, 5, 10, 15, and 20 μ g/L) in two-dimensional culture. On the other hand, bone marrow mesenchymal stem cells in the control group were induced with negative culture medium. Collagen type Ⅱ was qualitatively determined by using immunohistochemical method after two weeks, and glycosaminoglycan expression of extracellular matrix was quantitatively detected by using dimethyl-methylene-blue colorimetric method.MAIN OUTCOME MEASURES: Surface antigen, collagen type Ⅱ, and glycosaminoglycan expression of extracellular matrix.RESULTS: Bone marrow mesenchymal stem cells were separated and purified by using attachment culture method. The fourth-generated bone marrow mesenchymal stem cells were positive for surface antigen CD44, but negative for surface antigen CD34 and CD45. Cell form was irregular two weeks after induction. Immunohistocbemical staining on collagen type Ⅱ indicated that positive cells were observed in TGF- β 1 (1, 5, 10, 15, and 20 μg/L) groups. On the other hand, dimethyl-methylene-blue colorimetric method demonstrated that glycosaminoglycan expression of extracellular matrix in the TGF-β1 groups were significantly higher than in the control group (P < 0.01). In particular, glycosaminoglycan expression in the 10 μ g/L TOF- β 1 group was significantly higher than in other concentration groups (P < 0.01), and the capability of excreting glycosaminoglycan of extracellular matrix was positively correlated with cell densities (r=0.822, P < 0.01). CONCLUSION: A high cell density is beneficial to differentiation of bone marrow mesenchymal stem cells into chondrocytes induced by TGF-β1 (10 μg/L) in two-dimensional culture.