摘要
目的 探讨机械牵张通过Piezo1 促进小鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成骨分化的作用及机制.方法 将BMSCs分为 0%、3%、6%和 12%机械牵张强度组,CCK-8 检测细胞增殖,PCR检测成骨因子(Runx2、Osterix、ALP和OPN)mRNA,筛选出最佳牵张强度.再分别设置对照、机械牵张、Piezo1 抑制剂、机械牵张+Piezo1 抑制剂组.干预结束后,进行ALP染色和ALP 活性检测.茜素红染色观察钙结节形成.Western blot检测Piezo1、TGF-β1、p-Smad2 和Runx2 蛋白水平.结果 与 0%机械牵张相比,3%和 6%机械牵张促进了细胞增殖(P＜0.05),且 6%机械牵张组 Runx2、Osterix、ALP和OPN的mRNA均显著升高(P＜0.05).另外,与对照组相比,机械牵张组ALP染色较深,ALP活性显著升高,钙结节数量增加,Piezo1、TGF-β1、p-Smad2 和Runx2 蛋白也显著升高(P均＜0.05);Piezo1 抑制剂组ALP染色较浅,ALP活性显著降低,钙结节数量减少,Piezo1、TGF-β1、p-Smad2 和Runx2 蛋白也显著降低(P均＜0.05).与机械牵张组相比,机械牵张+Piezo1 抑制剂组ALP染色较浅,ALP活性显著降低,钙结节数量减少,Piezo1、TGF-β1、p-Smad2 和Runx2 蛋白也显著降低(P均＜0.05).与Piezo1 抑制剂组相比,机械牵张+Piezo1 抑制剂组ALP染色较深,ALP活性显著升高,钙结节数量增加,Piezo1、TGF-β1、p-Smad2 和Runx2 蛋白也显著升高(P均＜0.05).结论 机械牵张可能通过Piezo1 上调了TGF-β1/Smad2 信号通路表达,促进BMSCs的增殖和成骨分化.
Abstract
Objective To explore the role and mechanism of mechanical stretch in promoting osteogenic differentiation of mouse bone marrow mesenchymal stem cells(BMSCs)via Piezo1.Methods BMSCs were divided into 0%,3%,6%,and 12%mechanical stretch intensity groups.BMSCs were divided into 0%,3%,6%,and 12%mechanical stretch intensity groups.Cell proliferation was detected by CCK-8,and osteogenic factors(Runx2,Osterix,ALP,and OPN)mRNA were detected by PCR to screen for the optimal stretch intensity.Then,control,mechanical stretch,Piezo1 inhibitor,and mechanical stretch+Piezo1 inhibitor groups were set up separately.After the intervention,ALP staining and ALP activity testing were performed.Observe the formation of calcium nodules with alizarin red staining,and measure the OD570 nm value of each group after dissolving the calcium nodules.Piezo1 and TGF-β1,p-Smad2 and Runx2 protein levels were detected by Western blot.Results Compared with 0%mechanical stretch,3%and6%mechanical stretch promoted cell proliferation(P＜0.05),and the mRNA levels of Runx2,Osterix,ALP,and OPN were significantly increased in the6%mechanical stretch group(P＜0.05).In addition,compared with the control group,the mechanical stretch group showed deeper ALP staining,significantly increased ALP activity,number of calcium nodules,and the proteins of Piezo1,TGF-β1,p-Smad2 and Runx2(all P＜0.05);the Piezo1 inhibitor group showed lighter ALP staining,significantly reduced ALP activity,number of calcium nodules,and the proteins of Piezo1,TGF-β1,p-Smad2 and Runx2(all P＜0.05).Compared with the mechanical stretch group,the mechanical stretch+Piezo1 inhibitor group showed lighter ALP staining,significantly reduced ALP activity,number of calcium nodules,and the proteins of Piezo1,TGF-β1,p-Smad2 and Runx2(all P＜0.05).Compared with the Piezo1 inhibitor group,the mechanical stretch+Piezo1 inhibitor group showed deeper ALP staining,significantly increased ALP activity,number of calcium nodules,and the proteins of Piezo1,TGF-β1,p-Smad2 and Runx2(all P＜0.05).Conclusion Mechanical stretch may up-regulate the expression of the TGF-β1/Smad2 signaling pathway through Piezo1,and promotes the proliferation and osteogenic differentiation of BMSCs.
维普
万方数据