鸢尾素对大鼠骨髓间充质干细胞Sost基因表达及成骨分化的影响

Effect of Irisin on osteogenic differentiation and Sost of rat bone marrow mesenchymal stem cells

目的探究鸢尾素(Irisin)对大鼠骨髓间充质干细胞(BMSC)Sost基因表达及成骨分化的影响。方法设置Irisin不同浓度组(0、80、100、120ng/mL),细胞计数试剂盒(CCK-8)法选择Irisin的最佳实验浓度。用含有Irisin的培养液培养大鼠BMSC设置为实验组,对照组为单纯培养液培养,3、7、14d进行茜素红、vonKossa钙盐染色;3、10d采用反转录聚合酶链反应(RT-PCR)和Westernbolt检测成骨相关基因mRNA及蛋白表达。分别采用单因素方差分析和t检验对数据进行统计分析。结果(1)培养3d时,100ng/mL的Irisin促进大鼠BMSC增殖的效果明显(OD=0.951),与对照组相比差异有统计学意义(F=102.52,P<0.05);(2)实验组染色深度、钙结节大小和数量都明显高于对照组;(3)与对照组相比,实验组SostmRNA及其蛋白表达水平明显下降,ALP、Lrp5、BMP2、Smad1的mRNA及其蛋白表达水平明显增高,差异均有统计学意义(P<0.001)。结论(1)Irisin可抑制Sost基因的表达;(2)Irisin可在体外促进大鼠BMSC增殖和成骨分化;(3)Wnt和BMP信号通路可能在Irisin促进大鼠BMSC成骨分化中发挥作用。

Objective To investigate the effect of Irisin on osteogenic differentiation and Sost of rat bone marrow mesenchymal stem cells(BMSCs). Methods Irisin was set to have different concentrations (0,80,100,120 ng/mL),and the optimal concentration of Irisin was determined by CCK-8 method. The BMSCs cultured with Irisin was the experimental group,while the control group was cultured without Irisin. The alizarin red and von Kossa were stained on the 3rd,7th and 14th day to observe the positive region of osteogenic differentiation and the expression intensity. The mRNA and protein expression levels of the bone-related genes were detected by RT-PCR and Western bolt at 3rd and 10th days. The One-Way ANOVA and t -test were applied to perform the statistical analysis of the data. Results (1)Irisin at a concentration of 100 ng/mL promoted the proliferation of rat BMSCs significantly after cultured three days (OD=0.951;F =102.52,P<0.05).(2)The depth of staining and the size as well as the number of calcium nodules in the experimental group were significantly higher than those in the control group.(3)Compared with the control group,the mRNA and protein expression levels of the bone-related genes:ALP,Lrp5, BMP2 and Smad1 were significantly increased,but the expression levels of Sost mRNA and protein were significantly decreased(P<0.001). Conclusions (1)Irisin can inhibit the expression of the Sost gene (2)Irisin can promote the proliferation and osteogenic differentiation of rat BMSCs in vitro.(3)Wnt and BMP signaling pathway may play a role in Irisin promoting osteogenic differentiation of rat BMSCs.