酪蛋白激酶2相互作用蛋白1调控骨质疏松大鼠骨髓间充质干细胞的成骨能力

Casein kinase 2-interaction protein-1 regulates the osteogenic ability of bone marrow mesenchymal stem cells in osteoporosis rats

背景:目前对酪蛋白激酶2相互作用蛋白1(casein kinase 2-interaction protein-1,CKIP-1)分子机制的体外研究主要集中在基因敲除小鼠来源成骨细胞或骨髓间充质干细胞,鲜见报道骨质疏松模型大鼠来源骨髓间充质干细胞中CKIP-1表达的研究。目的:探讨下调CKIP-1基因前后骨质疏松状态骨髓间充质干细胞成骨分化能力的变化。方法:用维甲酸诱导雌性SD大鼠骨质疏松模型,采用全骨髓贴壁法体外培养骨质疏松组、正常组大鼠骨髓间充质干细胞。成骨诱导后进行茜素红染色、碱性磷酸酶染色及实时定量RT-PCR检测骨桥蛋白、Runx2 mRNA的相对表达,实时定量RT-PCR检测成骨诱导过程中2组细胞中CKIP-1的动态表达;通过基因转染沉默CKIP-1基因,成骨诱导后进行茜素红染色、碱性磷酸酶染色及实时定量RT-PCR检测骨桥蛋白、Runx2 mRNA的相对表达。结果与结论:①与正常组相比,骨质疏松组骨髓间充质干细胞茜素红染色钙结节定量、碱性磷酸酶活性及骨桥蛋白、Runx2的mRNA水平降低(P<0.05),骨质疏松组骨髓间充质干细胞中CKIP-1基因动态表达水平总体偏高;②与未下调CKIP-1的骨质疏松组骨髓间充质干细胞相比,下调CKIP-1基因表达后,骨质疏松组骨髓间充质干细胞茜素红染色钙结节定量、碱性磷酸酶活性及骨桥蛋白、Runx2的mRNA水平明显升高(P<0.05);③结果表明,维甲酸诱导的骨质疏松大鼠骨髓间充质干细胞成骨能力降低,下调CKIP-1基因可以部分提高其成骨分化能力。

BACKGROUND:At present,in vitro studies on the molecular mechanism of casein kinase 2-interaction protein-1(CKIP-1)mainly focus on gene knockout mouse-derived osteoblasts or bone marrow mesenchymal stem cells.There are few reports focusing on the expression of CKIP-1 in bone marrow mesenchymal stem cells derived from osteoporosis model rats.OBJECTIVE:To investigate the changes in osteogenic differentiation of bone marrow mesenchymal stem cells before and after down-regulation of CKIP-1 gene.METHODS:An osteoporotic rat model was induced by retinoic acid gavage.The bone marrow mesenchymal stem cells of the osteoporosis group and the normal group were cultured in vitro by the whole bone marrow adherence method.After osteogenic induction,Alizarin red staining,alkaline phosphatase staining and real-time quantitative RT-PCR were utilized to detect the relative expression of osteopontin and Runx2 mRNA.The dynamic expression of CKIP-1 in the two groups of cells was detected by using real-time quantitative RT-PCR.CKIP-1 gene was silenced by gene transfection.After osteogenic induction,alizarin red staining,alkaline phosphatase staining,and real-time quantitative RT-PCR were performed to detect the relative expression of osteopontin and Runx2 mRNA.RESULTS AND CONCLUSION:(1)Compared with the normal group,alizarin red staining of calcium nodules,alkaline phosphatase activity and mRNA levels of osteopontin and Runx2 in bone marrow mesenchymal stem cells decreased in the osteoporosis group(P<0.05).The dynamic expression level of CKIP-1 gene in bone marrow mesenchymal stem cells was generally higher in the osteoporosis group.(2)Compared with bone marrow mesenchymal stem cells of osteoporosis group without down-regulation of CKIP-1,after down-regulation of CKIP-1 gene expression,the quantification of calcium nodules and alkaline phosphatase activity by alizarin red staining,mRNA levels of osteopontin and Runx2 were significantly increased in bone marrow mesenchymal stem cells of the osteoporosis group(P<0.05).(3)Therefore,osteogenic ability of bone marrow mesenchymal stem cells in rats with retinoic acid-induced osteoporosis is reduced and down-regulation of CKIP-1 gene can partially improve their osteogenic differentiation ability.