降钙素基因相关肽调节滑膜间充质干细胞的增殖与成骨分化

Calcitonin gene-related peptide regulates proliferation and osteogenic differentiation of synovial mesenchymal stem cells

背景:已有研究证实降钙素基因相关肽可促进滑膜间充质干细胞的增殖与成骨分化,但是涉及其具体作用机制的研究较少。目的:观察降钙素基因相关肽干预对滑膜间充质干细胞增殖与成骨分化的影响,同时探讨Wnt/β-catenin信号通路在此过程中的作用。方法:采用胰蛋白酶联合Ⅰ型胶原酶消化法从兔膝关节滑膜组织中分离培养滑膜间充质干细胞。选择第3代滑膜间充质干细胞悬液,分4组培养:空白对照组常规培养,不进行任何干预;实验组加入10-8mol/L的降钙素基因相关肽;抑制剂组加入10-8mol/L的Wnt信号通路抑制剂DKK-1;联合干预组加入10-8mol/L的Wnt信号通路抑制剂DKK-1与10-8 mol/L的降钙素基因相关肽,干预14 d后进行相关指标检测。结果与结论:(1)抑制剂组β-catenin蛋白表达低于空白对照组(P <0.05),实验组β-catenin蛋白表达高于空白对照组(P <0.05),联合干预组β-catenin蛋白表达高于抑制剂组(P <0.05);(2)培养第3-9天,抑制剂组细胞增殖慢于其余3组(P <0.05);培养第5-9天,实验组细胞增殖始终快于空白对照组、联合干预组(P <0.05);(3)实验组碱性磷酸酶活性、矿化结节面积以及骨形态发生蛋白2、Runx2、碱性磷酸酶、Ⅰ型胶原基因表达高于其余3组(P <0.05),抑制剂组碱性磷酸酶活性、矿化结节面积以及骨形态发生蛋白2、Runx2、碱性磷酸酶、Ⅰ型胶原基因表达低于空白对照组(P <0.05),联合干预组碱性磷酸酶活性、矿化结节面积以及骨形态发生蛋白2、Runx2、碱性磷酸酶、Ⅰ型胶原基因表达高于抑制剂组(P <0.05);(4)实验证实降钙素基因相关肽能促进滑膜间充质干细胞的增殖与成骨分化,且Wnt/β-catenin信号通路与其增殖与成骨分化调控密切相关。

BACKGROUND:It has been proved that calcitonin gene-related peptide can promote the proliferation and osteogenic differentiation of synovial mesenchymal stem cells.However,little is reported on its specific mechanisms.OBJECTIVE:To investigate the effects of calcitonin gene-related peptide intervention on the proliferation and osteogenic differentiation of synovial mesenchymal stem cells,and to explore the role of Wnt pathway/β-catenin in this process.METHODS:Synovial mesenchymal stem cells were isolated from synovial tissues of the rabbit knee joint by trypsin and collagenase I digestion.The third generation suspension of synovial mesenchymal stem cells was selected and cultured in four groups.The blank control group was routinely cultured without any intervention.In the experimental group,10-8 mol/L calcitonin gene-related peptide was added.In the inhibitor group,10-8 mol/L DKK-1,a Wnt signal pathway inhibitor,was added.In the combined intervention group,10-8 mol/L calcitonin gene-related peptide and 10-8 mol/L DKK-1 were added.After 14 days of intervention,related indicator detections were performed.RESULTS AND CONCLUSION:(1)The expression ofβ-catenin protein in the inhibitor group was lower than that in the blank control group(P<0.05);the expression ofβ-catenin protein in the experimental group was higher than that in the blank control group(P<0.05);and the expression ofβ-catenin protein in the combined intervention group was higher than that in the inhibitor group(P<0.05).(2)The proliferation of cells in the inhibitor group was slower than that in the other three groups at 3-9 days of culture(P<0.05),while the proliferation of cells in the experimental group was always faster than that in the blank control group and combined intervention group at 5-9 days of culture(P<0.05).(3)The activity of alkaline phosphatase,mineralized nodule area and mRNA expression of bone morphogenetic protein 2,Runx2,alkaline phosphatase and type I collagen in the experimental group were higher than those in the other three groups(P<0.05).Compared with the blank control group,the inhibitor groups showed decreased alkaline phosphatase activity,reduced mineralized nodule area as well as reduced expression of bone morphogenetic protein 2,Runx2,alkaline phosphatase and type I collagen,while these indicators were higher in the combined intervention group than the inhibitor group(P<0.05).To conclude,intervention with calcitonin gene-related peptide can promote the proliferation and osteogenic differentiation of synovial mesenchymal stem cells through the Wnt/β-catenin signaling pathway.