低强度脉冲超声促进C2C12成肌细胞分化的作用机制研究  

The mechanism through which low-intensity pulsed ultrasound promotes the differentiation of C2C12 myoblasts

作　　者：张亚莲 舒彬 杨忠 李倩 蔡星 Zhang Yalian;Shu Bin;Yang Zhong;Li Qian;Cai Xing(Rehabilitation Center of University City Hospital Affiliated to Chongqing Medical University,Chongqing 401331,China;Department of Clinical Hematology,Army Military Medical University,Chongqing 400038,China)

机构地区：[1]重庆医科大学附属大学城医院康复中心,重庆401331 [2]陆军军医大学药学与检验医学系临床血液学教研室,重庆400038

出　　处：《中华物理医学与康复杂志》2021年第3期193-199,共7页Chinese Journal of Physical Medicine and Rehabilitation

基　　金：国家自然科学基金项目(31171148)。

摘　　要：目的观察不同功率密度低强度脉冲超声(LIPUS)对骨骼肌C2C12成肌细胞脂联素(Adiponectin)及其受体的影响,探讨LIPUS促进C2C12成肌细胞分化的潜在机制。方法将体外培养的C2C12成肌细胞根据有无超声干预或超声干预的功率密度随机分为对照组、U_(0.1)组、U_(0.3)组和U_(0.5)组,对照组接受假LIPUS干预,U_(0.1)组、U_(0.3)组、U_(0.5)组分别接受功率密度为0.1 W/cm^(2)、0.3 W/cm^(2)、0.5 W/cm^(2)的LIPUS干预。干预5 d后,分别采用CCK-8法检测细胞活力,采用荧光定量逆转录-聚合酶链反应(RT-PCR)法检测脂联素、脂联素受体1(AdipoR1)和T-钙黏蛋白(T-Cadherin)mRNA水平,采用Western blot法检测脂联素、AdipoR1、T-Cadherin、腺苷酸活化蛋白激酶(AMPK)、活化型-磷酸化腺苷酸活化蛋白激酶(P-AMPK)、肌细胞生成蛋白(MYOG)和胚胎肌球蛋白重链(eMHC)的蛋白水平,并对4组细胞肌管的分化能力进行免疫荧光化学检测。结果LIPUS干预5 d后,U_(0.1)、U_(0.3)和U_(0.5)组细胞相对活力明显高于对照组(P<0.01)。U_(0.3)组和U_(0.5)组脂联素、受体AdipoR1和T-Cadherin mRNA水平均显著上调,与对照组比较,差异均有统计学意义(P<0.05)。U_(0.3)组和U_(0.5)组脂联素、AdipoR1、T-Cadherin蛋白和AMPK磷酸化水平与对照组比较,均显著增加(P<0.05),且U_(0.1)组和U_(0.5)组均显著低于U_(0.3)组,差异均有统计学意义(P<0.01)。U_(0.3)组和U_(0.5)组C2C12成肌细胞分化指标eMHC、MYOG蛋白水平和C2C12成肌细胞融合指数与对照组比较,均明显增加,差异均有统计学意义(P<0.05)。结论LIPUS可促进C2C12成肌细胞的分化,并以0.3 W/cm^(2)、5 min/d、1 MHz,占空比为20%的LIPUS干预作用最明显,其调控机制可能与C2C12成肌细胞脂联素、受体AdipoR1和T-Cadherin的表达上调和下游AMPK磷酸化水平活化有关。Objective To observe the effect of low-intensity pulsed ultrasound(LIPUS)at different intensities on the expression of adiponectin and its receptors in C2C12 myoblasts,and to explore the potential mechanism by which LIPUS promotes the differentiation of C2C12 myoblasts.Methods C2C12 myoblasts cultured in vitro were randomly divided into a control group and U_(0.1),U_(0.3) and U_(0.5) groups.The control group received sham LIPUS exposure,while the U_(0.1),U_(0.3) and U_(0.5) groups were exposed to LIPUS at intensities of 0.1W/cm^(2),0.3W/cm^(2) or 0.5W/cm^(2) respectively,and 1MHz for 5 min daily for 5 days.Cell viability was measured using CCK-8 assays.Fluorescence quantitative reverse transcription-polymerase chain reactions were used to detect the mRNA expression of adiponectin,adiponectin receptor 1(adipoR1)and T-cadherin in the cells.Western blotting was employed to assess the protein expression of adiponectin,adipoR1,T-cadherin,adenosine monophosphate activated protein kinase(AMPK),activated phosphorylated adenylate-activated protein kinase(P-AMPK),embryonic myosin heavy chain(eMHC)and myogenin(MYOG).The differentiation ability of the 4 groups was measured using cell immunofluorescence chemistry.Results After the intervention the cell viability in the U_(0.1),U_(0.3) and U_(0.5) groups was significantly higher than in the control group.Compared with the control group,the average mRNA expression of adiponectin and the receptors of adipoR1 and T-cadherin were up-regulated significantly in the U_(0.3) and U_(0.5) groups.The average adiponectin,adipoR1 and T-cadherin protein expressions,and the AMPK phosphorylation level in the U_(0.3) and U_(0.5) groups had increased significantly compared with the control group,but all were significantly lower than in the U_(0.3) group.The average protein expression of eMHC and MYOG,and the C2C12 myoblast fusion indices of the U_(0.3) and U_(0.5) groups were significantly higher the control group′s averages.Conclusions LIPUS can promote the differentiation of C2C12 myoblasts

关 键 词：低强度脉冲超声波 脂联素 脂联素受体 分化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]