摘要
目的观察vaspin对体外培养SD大鼠骨髓间充质干细胞（ BMSCs）增殖及成骨分化的影响及其潜在机制。方法采用全骨髓法分离培养4周龄SD大鼠的BMSCs。成骨诱导液联合不同浓度vaspin （10、50、100 ng/ml）作用于BMSCs,分别于24、48、72 h采用CCK8法检测细胞增殖水平；于第7天用实时荧光定量聚合酶链反应（ RT-PCR）检测ALP、Runx2、β-catenin mRNA表达水平,Western印迹法检测β-catenin、Runx2蛋白表达水平。21天采用茜素红染色矿化结节及定量检测；在100 ng/ml vaspin中加入Wnt通路特异性阻断剂DKK1（1μg/ml）后,RT-PCR及Western印迹法再次检测上述指标表达水平。结果 Vaspin对大鼠BMSCs增殖无显著影响（P〉0.05）,可上调促进BMSCs成骨分化基因ALP、Runx2及β-catenin mRNA表达及β-catenin、Runx2蛋白表达,茜素红染色矿化结节呈红棕色且增多,定量检测OD值,vaspin组高于对照组（P〈0.05）。加入阻断剂DKK1后ALP、Runx2及β-catenin mRNA表达水平均显著下降（均P〈0.05）, Runx2及β-catenin蛋白表达水平亦显著下降。结论 Vaspin可通过Wnt/β-catenin信号通路促进BMSCs成骨分化。
Objective To observe the effect of vaspin on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells in rats and its potential mechanism. Methods Bone marrow mesenchymal stem cells（ BMSCs） from 4 weeks aged rats were isolated and cultured. The BMSCs were treated with osteogenic induction medium and different concentrations of vaspin and Wnt signaling pathway inhibitor DKK1. The proliferation was detected by CCK8 method at 24, 48, 72 h. After 7 days, the mRNA expressions of ALP, Runx2,β-catenin were detected by realtime qPCR. The expression levels of Runx2 and β-catenin protein were detected by Western blot. After 21 days, alizarin red stained mineralized nodules and quantitative detection were performed. Results Vaspin had no effect on the proliferation but promoted the expression of osteogenic differentiation gene ALP, Runx2, and also increasedβ-catenin mRNA and expression of Runx2 and β-catenin protein. Mineralized nodules were brown and increased, OD value of vaspin group was higher than control group. After adding the DKK1, the expression of ALP, Runx2,β-catenin mRNA and Runx2, β-catenin protein were significantly decreased（all P〈0. 05）. Conclusion Vaspin can promote BMSCs into osteogenic differention through the Wnt/β-catenin signaling pathway.
出处
《中华内分泌代谢杂志》
CAS
CSCD
北大核心
2016年第6期505-509,共5页
Chinese Journal of Endocrinology and Metabolism
基金
基金项目：山西省科技攻关项目（20130313022-4）
