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

doi:10.3969/j.issn.2095-4344.0667

Abstract

Abstract:

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 of β-catenin protein in the inhibitor group was lower than that in the blank control group (P < 0.05); the expression of β-catenin protein in the experimental group was higher than that in the blank control group (P < 0.05); and the expression of β-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.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Synovial Membrane, Mesenchymal Stem Cells, Calcitonin Gene-Related Peptide, Cell Proliferation, Cell Differentiation, Tissue Engineering

CLC Number:

R394.2

Deng Rui, Pan Fu-wen, Han Yao-guang. Calcitonin gene-related peptide regulates proliferation and osteogenic differentiation of synovial mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(33): 5321-5326.

Figures/Tables 1

2.1 滑膜间充质干细胞的培养与鉴定结果采用倒置显微镜观察发现，原代培养的细胞在1 d之后开始贴壁；2 d后可见大量形态各异的细胞，多呈圆形或多角形；三四天后，细胞融合可达80%左右，呈漩涡状排列；传代第3代后，细胞形态逐渐均一，呈梭形，见图1；随着传代次数的增加，细胞形态无明显变化。选择第3代滑膜间充质干细胞进行流式细胞仪鉴定，发现其表面可高表达CD90、CD105，低表达CD68。 2.2 Western blotting检测β-catenin蛋白表达与空白对照组比较，抑制剂组β-catenin蛋白表达明显降低，差异有显著性意义(P < 0.05)；与空白对照组比较，实验组β-catenin蛋白表达明显升高，差异有显著性意义(P < 0.05)；与抑制剂组比较，联合干预组β-catenin蛋白表达明显升高，差异有显著性意义(P < 0.05)，见图2。 2.3 CCK-8法检测细胞增殖随着培养时间的增加，各组细胞吸光度值逐渐增加；培养第3天，抑制剂组细胞吸光度值低于其余各组，差异有显著性意义(P < 0.05)，其余组间比较差异无显著性意义；培养第5-9天，抑制剂组细胞吸光度值低于空白对照组，差异有显著性意义(P < 0.05)，实验组细胞吸光度值高于空白对照组、联合干预组，差异有显著性意义(P < 0.05)，联合干预组细胞吸光度值高于抑制剂组，差异有显著性意义(P < 0.05)，见图3。 2.4 茜素红染色结果成骨诱导分化14 d，茜素红染色观察滑膜间充质干细胞矿化结节生成情况。结果显示各组均有呈橘红色的矿化结节生成(图4)，实验组矿化结节最多且最大，抑制剂组矿化结节最少且最小，空白对照组矿化结节较联合干预组多且大；采用ImageJ软件分析染色面积，实验组矿化结节面积大于其余3组，差异有显著性意义(P < 0.05)，空白对照组矿化结节面积大于抑制剂组、联合干预组，差异有显著性意义(P < 0.05)，联合干预组矿化结节面积大于抑制剂组，差异有显著性意义(P < 0.05)。 2.5 碱性磷酸酶活性成骨诱导分化14 d，实验组碱性磷酸酶活性高于空白对照组、联合干预组、抑制剂组，差异有显著性意义(P < 0.05)；与空白对照组比较，抑制剂组碱性磷酸酶活性下降，差异有显著性意义(P < 0.05)；与抑制剂组比较，联合干预组碱性磷酸酶活性升高，差异有显著性意义(P < 0.05)，见图5。 2.6 RT-PCR检测成骨相关基因表达成骨诱导分化14 d，RT-PCR检测成骨相关基因碱性磷酸酶、骨形态发生蛋白2、Runx2及Ⅰ型胶原基因表达。结果可见，实验组碱性磷酸酶、骨形态发生蛋白2、Runx2及Ⅰ型胶原基因表达高于空白对照组、联合干预组、抑制剂组，差异有显著性意义(P < 0.05)；抑制剂组碱性磷酸酶、骨形态发生蛋白2、Runx2及Ⅰ型胶原基因表达明显低于空白对照组，差异有显著性意义(P < 0.05)；联合干预组碱性磷酸酶、骨形态发生蛋白2、Runx2及Ⅰ型胶原基因表达明显高于抑制剂组，差异有显著性意义(P < 0.05)，见图6。"

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