鸢尾素对静态和牵张力作用下BMSCs成骨向分化的研究

Effect of irisin on osteogenic differentiation of bone marrow mesenchymal stem cells under static force and mechanical strain

摘要:

目的：在静态及牵张力作用下，研究鸢尾素（irisin）对大鼠骨髓间充质基质细胞（bone marrow mesenchymal stromal cells，BMSCs）成骨向分化的影响。方法：分离培养原代大鼠BMSCs，应用四点弯曲加力系统建立体外细胞牵张模型。通过碱性磷酸酶（alkaline phosphatase，ALP）活性检测在静态及牵张力作用下，不同浓度的鸢尾素对BMSCs成骨向分化的影响并筛选最适鸢尾素浓度。实验分组如下：A，对照组；B，鸢尾素组（100 ng/mL鸢尾素）；C，牵张力组（2 000 με牵张力）；D，鸢尾素+牵张力组（100 ng/mL 鸢尾素+2 000 με 牵张力）。细胞计数法检测各组细胞增殖情况，通过定量逆转录PCR（qRT-PCR）及Western blot检测成骨相关mRNA及蛋白的表达，并检测P38、ERK1/2 MAPK通路表达。采用SPSS 20.0软件对数据进行统计学分析。结果：ALP活性检测结果示，在静态及牵张力作用下，鸢尾素浓度为100 ng/mL时，BMSCs的ALP活性显著升高（P<0.01）。细胞增殖检测结果示，与对照组相比，鸢尾素+牵张力组可促进BMSCs的增殖（P<0.05）。qRT-PCR结果示，鸢尾素+牵张力可促进成骨标志物Runx2、ALP、 COL-Ⅰ、OPN表达的升高（P<0.05）。Western blot结果示，鸢尾素+牵张力可促进Runx2蛋白表达的升高（P<0.05）。此外，鸢尾素+牵张力联合作用促进BMSCs增殖与成骨向分化的机制可能与P38、ERK1/2 MAPK通路的激活有关。结论：鸢尾素对静态和体外牵张力作用下BMSCs增殖及成骨向分化具有促进作用，此效应可能通过P38、ERK1/2 MAPK通路发挥作用。

Abstract:

Objective：To investigate the effect of irisin on the osteogenic differentiation of rat bone marrow mesenchymal stem cells （BMSCs） under static force and mechanical strain. Methods：Primary rat BMSCs were isolated and cultured，and a four-point bend－ing force system was used to establish an in vitro model of cell stretch. The activity of alkaline phosphatase（ALP） was measured to analyze the effect of irisin concentration on the osteogenic differentiation of BMSCs under static force and mechanical strain and screen out the optimal concentration of irisin. BMSCs were divided into control group，irisin group（100 ng/mL irisin），mechanical strain group（2 000 με mechanical strain），and irisin+mechanical strain group（100 ng/mL irisin+2 000 με mechanical strain）. Cell counting was performed to observe cell proliferation，qRT-PCR and Western blot were used to measure the mRNA and protein expression of osteogenesis-related markers，and the expression of the P38 and ERK 1/2 MAPK pathways was measured. SPSS 20.0 software was used for statistical analysis. Results：The results of ALP activity showed that under static force and mechanical strain，BMSCs treated with 100 ng/mL irisin had a significant increase in ALP activity（P<0.01）. Cell proliferation assay showed that compared with the control group，the irisin+mechanical strain group had a significantly higher level of proliferation of BMSCs（P<0.05）. The results of qRT-PCR showed that irisin + mechanical strain significantly promoted the expression of os－teogenesis-related markers including Runx2，ALP，COL-I，and OPN（P<0.05），and Western blot showed that irisin+mechanical strain significantly promoted the protein expression of Runx2（P<0.05）. In addition，irisin+mechanical strain promoted the proliferation and osteogenic differentiation of BMSCs，which might be associated with the activation of the P38 and ERK 1/2 MAPK pathways. Conclusion：Irisin can promote the proliferation and osteogenic differen－tiation of BMSCs under static force and mechanical strain，possibly via the P38 and ERK1/2 MAPK pathways.