胰岛素样生长因子-1和转化生长因子-β3对人肌腱细胞表型分化标志物mRNA表达的影响

Effects of insulin-like growth factor-1 and transforming growth factor-β3 on tenocyte＇s phenotype and the mRNA expression of tenocyte phenotypic and differentiation markers

目的 观察在无血清条件下,不同浓度的胰岛素样生长因子（IGF）-1和转化生长因子（TGF）-β3在人肌腱细胞体外培养中对人肌腱细胞的表型及其标志物基因表达的影响.方法 在不添加胎牛血清（FBS）的α-MEM培养基中,加入IGF-1 （50μg/L）、TGF-β3（10 μg/L）以及IGF-1（50 μg/L）＋ TGF-β3（10μg/L）,同时使用10％的FBS作为阳性对照组,用天狼猩红染色细胞观察细胞第14天的表型变化,同时在细胞培养第14天提取各组细胞mRNA,采用实时定量逆转录聚合酶链反应（RT-qPCR）测定人肌腱细胞表型及分化标志物Scleraxis、Tenomodulin、Ⅰ型胶原蛋白（Collagen Ⅰ）以及核心蛋白聚糖（Decorin）的基因表达.结果 在没有添加FBS的条件下,各实验组及对照组细胞形态均未出现表型变异,与阳性对照组的细胞比较,IGF-1（50 μg/L）＋TGF-β3（10 μg/L）组的细胞形态呈现出较强分化状态,有较多的胶原产生,同时胶原纤维的排列浓密有序并呈现出平型排列的胶原纤维形态,与阳性对照组极为类似.与阳性对照组比较,IGF-1（50tμg/L）＋TGF-β3（l0 μg/L）组的肌腱细胞表型标志物的基因表达比值明显上调（Scleraxis为132.5,=3.35,P＜0.01;Tenomodulin为536.3,t=115.89,P＜0.01;Collagen Ⅰ为5.22,t =5.94,P＜0.01及Decorin为1.40,t=5.67,P＜0.05）,差异有统计学意义.结论 在不使用FBS的α-MEM培养基中添加IGF-1（50 μg/L）＋TGF-β3（10μg/L）可维持人肌腱细胞表型,胶原纤维产生类似于添加10％ FBS培养的肌腱细胞,同时上调肌腱细胞的表型及分化标志物的mRNA表达.

Objective To evaluate the tenocyte phenotype changes and the expression of the phenotypic markers by adding insulin-like growth factor （IGF）-1 and transforming growth factor （TGF）-β3 to human tenocyte in vitro culture.Methods Human tenocytes were cultured in fatal bovine serun （FBS）-free α-MEM medium supplemented with IGF-1 and TGF-β3.Sirius red staining was employed to evaluate the characteristics of the tenocytes cultured.Real-time reverse transcriptase-polymerase chain reaction （RT-qPCR） technique was used to detect the mRNA expression of the tenocyte phenotypic and differentiation markers.Results The tenocytes cultured for 14 days with 0% FBS,IGF-1 （50 μg/L） and TGF-β3 （10 μg/L） showed similar phenotypic characteristics in comparison with those cultured in 10% FBS.The tenocytes cultured in the treated group also showed up-regulated mRNA expression of those markers （for scleraxis,132.5,t =3.35,P 〈0.01 ; for tenomodulin,536.3,t =115.89,P 〈0.01 ; for collagen I,5.22,t=5.94,P〈0.01; forDecorin,1.40,t=5.67,P〈0.05）.Conclusion These findings suggest that human tenocytes could maintain their phenotype and tenocyte differentiation could be promoted in the FBS-free culture media containing IGF-1 （50 μg/L） and TGF-β3 （10 μg/L）.