机械牵张力促进小鼠骨髓间充质干细胞的成骨向分化

摘要

Objective This study aimed to investigate the stimulating mechanism of p38 mitogen-activated protein kinase(MAPK) signaling pathway and Osterix under intermittent mechanical stretch tension to accelerate the osteogenic differentiation of bone mesenchymal stem cells(BMSCs) in mice.Methods Three groups of C57BL/6J BMSC,namely,blank control group,strain group,and inhibitor group(p38MAPK signal pathway inhibitor SB203580 + strain),were prepared and exposed to 0.8％ intermittent mechanical strain at 0.5 Hz twice a day for 30 min at each time by using a Flexercell strain unit.The cells in the three groups were then harvested on days 1,3,and 5,respectively.Changes in the mRNA expression of ALP,COL Ⅰ,and OCN were detected through real time-polymerase chain reaction(RT-PCR) and the protein expression of P-p38MAPK was observed through Western blot analysis.osterix gene was knocked down by small interfering RNA(siRNA),and Osterix protein expression was determined through Western blot.The mRNA expression levels of ALP,COL Ⅰ,and OCN were identified through RT-PCR.Results Mechanical tension force could promote the mRNA expression of ALP,COL Ⅰ,OCN,and osterix.osterix silencing could decrease the mRNA expression of ALP,COL Ⅰ,and OCN.Western blot revealed that the protein expression levels of Osterix and P-p38MAPK in the strain group were significantly higher than those in the control group(P＜0.05).The mRNA expression of ALP,COL Ⅰ,OCN,and osterix decreased after SB203580 was used.Conclusion Intermittent mechanical strain promotes the osteogenic differentiation of BMSC via the p38MAPK-Osterix pathways.%目的 旨在研究p38分裂原激活的蛋白激酶(MAPK)信号通路和转录因子Osterix在间断性机械牵张力刺激下促进小鼠骨髓间充质干细胞(BMSC)成骨向分化的作用机制.方法 将C57BL/6J小鼠BMSC分为空白对照组、牵张力组和牵张力阻断组(p38MAPK通路抑制剂SB203580+牵张力),采用Flexercell体外细胞力学加载装置,施加频率为0.5 Hz、形变率为0.8％的牵张力,每天2次,每次30 min,分别在实验第1、3、5d收获BMSC.实时荧光定量聚合酶链反应(RT-PCR)检测成骨基因ALP、COLⅠ、OCN的mRNA水平变化情况,Western blot检测P-p38-MAPK蛋白的表达情况.通过小干扰核糖核酸(siRNA)技术沉默小鼠BMSC的osterix基因,Westem blot检测Osterix蛋白的表达情况,RT-PCR检测成骨相关基因ALP、COLⅠ、OCN的mRNA水平变化情况.结果 牵张力作用后,可观察到成骨相关基因ALP、COLⅠ、OCN及转录因子Osterix的mRNA水平增高.沉默osterix后,小鼠BMSC成骨相关基因ALP、COLⅠ、OCN的mRNA水平也随之降低.Western blot结果显示,牵张力组小鼠BMSC中Osterix和P-p38-MAPK的蛋白质水平明显高于对照组(P＜0.05).SB203580作用后,小鼠BMSC成骨相关基因ALP、COLⅠ、OCN和osterix的mRNA水平降低.结论 间断性牵张力可通过活化p38MAPK-Osterix通路促进小鼠BMSC成骨分化.