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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β1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1,5,10,15,20μg/L的转化生长因子β1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,15,10,15,20μg/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)。10μg/L转化生长因子β1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:10μg/L转化生长因子β1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。
BACKGROUND: Bone marrow mesenchymal stem cells can be induced to chondrocytes. OBJECTIVE: To establish an induction system of MSCs differentiating into chondrocytes under two-dimensional culture conditions, analyze the optimal concentration of transforming growth factor-β1 as inducing condition, and the related factors that induce the differentiation of MSCs into chondrocytes , Observe the cell morphology and phenotype changes induced by cells. Design: Randomized controlled animal experiments. Unit: Animal Experiment Center of Guiyang Medical College. MATERIALS: Experiments were performed at Animal Experimental Center of Guiyang Medical College from September 2005 to November 2006. 24 male Sprague-Dawley rats, 4 weeks old, were provided by Animal Center of Guiyang Medical College and their body weight was 98.23 ± 7.97 g. The animals were treated in accordance with animal ethical standards during the experiment. Methods: Bone marrow and tibia of SD rats were isolated and purified by adherent culture. BMSCs of 4th generation were identified by flow cytometry. , 5,10,15,20μg / L of transforming growth factor-β1 induction medium on the second generation of culture conditions on the fourth generation of bone marrow mesenchymal stem cells cultured in the control group was added to the negative induction medium. Two weeks later, type Ⅱ collagen was detected by immunohistochemical method, and the expression of extracellular matrix glycosaminoglycan was quantitatively detected by dimethyl-methylene blue colorimetry. MAIN OUTCOME MEASURES: Cell surface antigen detection, type Ⅱ collagen qualitative detection and extracellular matrix glycosaminoglycan expression detection in each group. Results: Adherent culture method could be used to isolate and purify bone marrow mesenchymal stem cells from SD rats, and the fourth generation BMSCs surface antigen CD44 positive, CD34 and CD45 negative. After 2 weeks of induction, the cell morphology became irregular. Immunohistochemical staining of type Ⅱ collagen showed that 15, 15, 15, 15, 20 μg / L transforming growth factor β1 group showed positive cells. Methyl methylene blue colorimetric assay The contents of extracellular matrix glycosaminoglycans in each experimental group were significantly higher than those in the control group (P <0.01). The amount of extracellular matrix glycosaminoglycan secreted by 10μg / L TGFβ1 group was significantly more than other dose groups (P <0.01), and the ability of cells to secrete extracellular matrix glycosaminoglycan was positively correlated with cell density (r = 0.822, P <0.01). CONCLUSION: Higher cell density in the presence of 10μg / L transforming growth factor β1 is beneficial to differentiation of BMSCs into chondrocytes.