转化生长因子-?茁1促进骨形态发生蛋白9诱导间充质干细胞成骨分化

Promotion of bone morphogenetic protein 9-induced mesenchymal stem cell osteogenesis by transforming growth factor-?茁1

摘要:

目的：探讨转化生长因子?茁1（transforming growth factor-?茁1，TGF-?茁1）在骨形态发生蛋白9（bone morphogenetic protein 9，BMP9）诱导间充质干细胞（mesenchymal stem cell，MSC）成骨分化过程中的作用。方法：应用鼠MSC C3H10T1/2，以重组腺病毒法将BMP9导入，施加TGF-?茁1处理，建立BMP9联合TGF-β1诱导MSC成骨分化模型。改良SEAP化学发光法检测碱性磷酸酶（alkaline phosphatase，ALP）活性；茜素红S染色进行钙盐沉积检测；实时荧光定量PCR检测成骨相关基因Ⅰa2型胶原（collagenⅠa2，COLⅠa2）、骨桥素（osteopotin，OPN）、骨钙素（osteocalcin，OCN）的mRNA转录表达水平，免疫细胞化学法检测细胞内OPN、OCN表达变化。荧光素酶报告基因法检测信号通路的Smad水平。结果：TGF-?茁1与BMP9联合处理较BMP9单独处理能更早更多地提高ALP活性和引起钙盐沉积（F处理=18 782.10，P=0.000），持续处理达17 d后不再显示明显差异，TGF-?茁1单独处理与对照组间无明显差异（F=1.03，P=0.318）。TGF-?茁1与BMP9联合处理较BMP9单独处理可使COLⅠa2、OPN、OCN mRNA转录水平明显增高（COLⅠa2的F处理=250.30，P=0.000；OPN 的F处理=795.64，P=0.000；OCN的F处理=206.55，P=0.000），COLⅠa2增高发生较早和显著（F=250.30，P=0.000）；细胞内OPN、OCN蛋白阳性表达显著增强。TGF-β1联合BMP9处理能刺激信号通路相应的Smad荧光素报告活性（?字2=32.84，P=0.000）。结论：在BMP9诱导MSC成骨分化中，联合应用TGF-?茁1具有促进成骨作用，BMP9与TGF-?茁1在成骨分化中可能存在互补效应。

Abstract:

Objective：To investigate the effect of transforming growth factor-β1（TGF-β1） on bone morphogenetic protein 9（BMP9）-induced mesenchymal stem cells（MSCs） osteogenic differentiation. Methods：C3H10T1/2 cell line infected with BMP9 adenovirus and added with TGF-β1 protein was used to create the osteogenic model. The modified SEAP chemoluminescence assay was used for al－kaline phosphatase（ALP） activity. Alizarin red staining was used for mineralization. Real-time PCR and immunocytochemistry were used for bone markers collagen typeⅠa2（COLⅠa2），osteopotin（OPN） and osteocalcin（OCN）. Luciferase reporter assay was used for detecting the Smad expression. Results：BMP9 combined with TGF-β1 induced more ALP activities and mineralization（F=18 782.10，P=0.000） than single BMP9. However，there was no difference between single TGF-β1 group and single GFP group after treating con－secutively for 17 d（F=1.03，P=0.318）. mRNA expression of OPN，OCN and COLⅠa2 was increased in BMP9 combined with TGF-β1 group than in single BMP9 group（FCOLⅠa2=250.30，P=0.000；FOPN=795.64，P=0.000；FOCN=206.55，P=0.000）. BMP9 combined with TGF-β1 increased COLⅠa2 more significantly at earlier stage（F=250.30，P=0.000） and increased the protein expression of OPN and OCN in vitro. BMP9 combined with TGF-β1 stimulated the activity of luciferase reporter of related Smads（?字2=32.84，P=0.000）. Conclu－sions：TGF-β1 promotes BMP9-induced osteogenic differentia－tion in MSCs. TGF-β1 and BMP9 might have synergistic effects.