骨髓间充质干细胞在β3转化生长因子联合胰岛素样生长因子-1定向诱导下的软骨组织工程

摘要

目的 探讨β3转化生长因子(TGF-β3)在诱导骨髓间充质干细胞(mesenchymal stem cells,MSCs)向软骨细胞分化中胰岛素样生长因子-1(IGF-1)的作用以及在软骨组织工程中的应用. 方法 在体外用TGF-β3或(和)IGF-1诱导藻酸钠微球巾的MSCs向软骨细胞定向分化,免疫组织化学、逆转录-聚合酶链反应(RT-PCR)检测Ⅱ型胶原和聚集蛋白聚糖(aggrecan)的表达,Western印迹法检测Sox9蛋白的表达,激光共聚焦显微镜和扫描电镜观察该软骨细胞在壳聚糖支架上的生长.结果 TGF-β3能诱导藻酸钠微球中的MSCs表达软骨特异性的Ⅱ型胶原、聚集蛋白聚糖和Sox9,IGF-1能显著性地增强这种作用(P＜0.05).Ⅱ型胶原、聚集蛋白聚糖和Sox9之间的相关系数分别为0.95和0.91.诱导的软骨细胞能在壳聚糖支架上黏附、迁徙、增殖.结论在TGF-β3诱导MSCs分化成软骨细胞地过程中,IGF-1可能通过促进Sox9的表达起到协同作用.诱导分化后的软骨细胞与壳聚糖复合支架表现出良好的组织相容性.%Objective To investigate the effect of IGF-1 on the TGF-β3 induced chondrogenesis of MSCs encapsulated in alginate beads and its application in cartilage tissue engineering.Methods MSC chondrogenesis in alginate beads was induced by TGF-β3 and/or IGF-1.Collagen type Ⅱ,aggrecan and Sox-9 expression was evaluated by immunostaining,RT-PCR and Western blot,respectively.Scanning electron microscope and laser confocal microscope were used to observe the differentiated chondrocytes when cultured on the chitosan-based scaffold.Results TGF-β3 with IGF-1 induced MSCs in alginate beads to express the higher level of collagen type Ⅱ,aggrecan and Sox-9 than any other growth factor alone (P＜0.05).The correlation coefficient between Sox9 and collagen type Ⅱ or aggrecan was 0.95 and 0.91,respectively.The chitosan-based scaffold supported the cell's adhesion,migration and proliferation.Conlusion IGF-1 enhances the TGF-β3-induced MSC chondrogenesis via upregulating Sox9 expression.The chitosan-based scaffold is biocompatible with the differentiated chondrocytes.