体外诱导骨髓间充质干细胞定向分化为软骨细胞:转化生长因子β1、胰岛素样生长因子1的协同刺激作用

Optimization of the protocols for induction and differentiation of bone marrow mesenchymal stem cells into chondrocytes: a synergistic action between transforming growth factor beta1 and insulin-like growth factor 1

背景:目前诱导骨髓间充质干细胞分化为软骨细胞主要以使用诱导因子的方法为主。目的:分析转化生长因子β1、胰岛素样生长因子1体外诱导骨髓间充质干细胞定向分化为软骨细胞的协同刺激作用,以求获得最佳的诱导效应。方法:取SD大鼠骨髓,采用全骨髓培养+贴壁纯化法分离培养骨髓间充质干细胞。按添加诱导因子不同分4组:转化生长因子β1+胰岛素样生长因子1组、转化生长因子β1组、胰岛素样生长因子1组、对照组。在基础诱导液基础上添加相应诱导因子诱导骨髓间充质干细胞向软骨细胞分化。诱导21 d后行Ⅱ型胶原免疫荧光染色,诱导7,14,21 d后行免疫细胞化学染色检测Ⅱ型胶原表达。结果与结论:(1)诱导21 d后,转化生长因子β1+胰岛素样生长因子1组、转化生长因子β1组免疫荧光显示胞浆呈棕红色,高度表达Ⅱ型胶原,而胰岛素样生长因子1组和对照组结果呈阴性;(2)免疫细胞化学染色显示,转化生长因子β1+胰岛素样生长因子1组在各个时间段的Ⅱ型胶原表达量明显高于转化生长因子β1组(P<0.01),且随着时间的延长,表达量逐渐增加。胰岛素样生长因子1组和对照组免疫细胞化学染色呈阴性;(3)结果表明,体外诱导骨髓间充质干细胞分化为软骨细胞时,转化生长因子β1与胰岛素样生长因子1具有协同刺激作用,二者联合使用可获得较佳的诱导效应。

BACKGROUND： Inducing factors are currently used as a main method for the differentiation of bone marrow mesenchymal stem cells（BMSCs） into chondrocytes. OBJECTIVE： To investigate the collaborative stimulation of transforming growth factor beta1（TGF-β1） and insulin-like growth factor 1（IGF-1） to induce the directed differentiation of BMSCs to chondrocytes, and to explore the best inductive effect. METHODS： Rat BMSCs were isolated, cultured and purified using adherent culture. Then, different inducing factors were added in the induction medium： TGF-β1＋IGF-1 group, TGF-β1 group, IGF-1 group, and control group without growth factors. Immunofluorescence was carried out at 21 days of induction. The expression of collagen type II was evaluated by immuncytochemical staining at 7, 14 and 21 days of induction. RESULTS AND CONCLUSION：（1） Immunofluorescence detection of the TGF-β1＋IGF-1 and TGF-β1 groups showed highly expressed collagen type II（brown red-stained cytoplasm）, while negatively expressed collagen type II in the other two groups.（2） Findings from the immuncytochemical staining showed that the expression of collagen type II was stronger in the TGF-β1＋IGF-1 group than the TGF-β1 group（P〈0.01）, and the expression was gradually enhanced with time. Meanwhile, there was also no expression of collagen type II in the IGF-1 and control groups. To conclude, the combination of TGF-β1 and IGF-1 can achieve the better inductive effect on the chondrogenic differentiation of BMSCs in vitro.