机械牵拉促进小鼠骨骼肌卫星细胞增殖并抑制成骨成脂分化的作用机制研究  

Effects and mechanism of mechanical stretch promoting proliferation and inhibiting osteogenic and adipogenic differentiation of skeletal satellite cells in mice

作　　者：高招文 李宏云 吴建斌 张淑欣 叶剑 吴冯春 Gao Zhaowen;Li Hongyun;Wu Jianbin;Zhang Shuxin;Ye Jian;Wu Fengchun(Department of Orthopaedics,The First Hospital of Nanping,Affiliated to Fujian Medical University,Nanping 353000,China;Department of Sports Medicine,Huashan Hospital,Fudan University,Shanghai 200040,China)

机构地区：[1]福建医科大学附属南平第一医院骨关节科,福建南平353000 [2]复旦大学附属华山医院运动医学科,上海200040

出　　处：《中国运动医学杂志》2024年第8期637-646,共10页Chinese Journal of Sports Medicine

基　　金：福建省自然科学基金项目(2021J011426)。

摘　　要：目的:研究机械牵拉对小鼠骨骼肌卫星细胞增殖和成骨成脂分化的影响并明确其作用机制。方法:本实验共分为两大部分,第一部分在未进行成骨及成脂诱导条件下进行,主要对细胞增殖活性和迁移能力进行评估,并检测机械牵拉对Wnt通路和Notch信号通路的影响,实验共分为五组:(1)空白对照组;(2)机械牵拉组;(3)溶媒对照组(二甲基亚砜(DMSO)+机械牵拉);(4)Notch抑制剂(KY-02111)+机械牵拉组;(5)Wnt抑制剂(FLI-06)+机械牵拉组。第二部分在进行成骨及成脂诱导条件下进行,实验共分为六组:(1)空白对照组;(2)诱导分化组;(3)诱导分化+机械牵拉组;(4)诱导分化+DMSO+机械牵拉组;(5)诱导分化+Notch通路抑制剂(KY-02111)+机械牵拉组;(6)诱导分化+Wnt通路抑制剂(FLI-06)+机械牵拉组。空白对照组小鼠骨骼肌卫星细胞不做处理,诱导分化组分别使用成骨或成脂诱导培养基培养细胞,诱导分化+机械牵拉组在细胞诱导培养的同时对细胞基底膜施加牵拉应力,诱导分化+Notch通路抑制剂(KY-02111)/Wnt通路抑制剂(FLI-06)+机械牵拉组在诱导培养和机械牵拉的同时分别向细胞培养液中添加终浓度为5μM的KY-02111或者FLI-06,诱导分化+DMSO+机械牵拉组向细胞培养液中添加与通路抑制剂等体积的DMSO作为溶媒对照。于培养后的第7和14天,CCK-8法检测细胞增殖活性。培养后的第14天,划线法检测各组细胞迁移能力;对细胞进行碱性磷酸酶和油红染色;通过实时荧光定量检测成骨分化标志基因碱性磷酸酶(ALP)、Runt相关转录因子(Runx)-2以及成脂分化标志基因脂肪酸结合蛋白(FABP)4和脂蛋白脂肪酶(LPL)的m RNA表达变化;免疫印迹检测c-Myc和细胞周期蛋白B (CCNB)1蛋白表达。结果:(1)细胞增殖活性检测结果表明,与对照组比较,机械牵拉组骨骼肌卫星细胞的增殖活性明显增强(P<0.01);与机械牵拉组比较,Notch和Wnt通路抑制剂+机械牵拉组细胞增殖�Objective To investigate the effect of mechanical stretch on the proliferation and differentiation of skeletal satellite cells in mice,and explore its mechanism.Methods The research was divided into two parts.In the first part,all mice were not given osteogenic or adipogenic induction,and randomly divided into a control group,a mechanical stretch(MS)group,a solvent control group[Dimethyl sulfoxide(DMSO)+mechanical stretch],a Notch inhibitor(KY-02111)+mechanical stretch group and a Wnt inhibitor(FLI-06)+mechanical stretch group,with the aim to evaluate cell proliferation and migration,and the effect of mechanical stretch on the Wnt and Notch signaling pathway.In the second part,all mice were randomly divided into 6 groups:a control group,an induced differentiation(ID)group,an induced differentiation+mechanical stretch(IDMS)group,an induced differentiation+DMSO+mechanical stretch(IDDMS)group,an induced differentiation+Notch inhibitor(KY-02111)+mechanical stretch(IDNMS)group,and an induced differentiation+Wnt inhibitor(FLI-06)+mechanical stretch(IDWMS)group.All groups except the control group were induced differentiation using osteoblastic or adipogenic medium,and the IDMS group was applied tensile stress to the cell basement membrane,with the IDDMS and IDNMS groups given DMSO and KY-02111 or FLI-06with a final concentration of 5μM into the culture medium,respectively.On the 7^(th) and 14^(th) days after culture,cell proliferation was detected using CCK-8,while on the 14thday after culture,cell migration was observed using the streak method.Alkaline phosphatase and oil red staining were performed on all cells.Moreover,the mRNA expression of osteogenic[alkaline phosphatase(ALP)and Runt-related transcription factor(Runx)-2]and adipogenic[fatty acid-binding protein(FABP)4 and lipoprteinlipase(LPL)]differentiation marker genes were quantitatively detected by using the real-time PCR.Meanwhile,while the expression of c-Myc and Cyclin B(CCNB)1 proteins was measured using Western blotting.Results Cell proliferation increas

关 键 词：骨骼肌卫星细胞 分化 机械牵拉 WNT信号通路 NOTCH信号通路 

分 类 号：R685.4[医药卫生—骨科学]