不同频率周期性应力加载对体外多层肌管极性及分化的影响

目的探讨不同频率的周期性应力加载对体外培养多层肌管极性与分化的影响,筛选优化的肌组织体外应力加载培养条件。方法体外培养C2C12成肌细胞于Sylgard 184铸型凹槽诱导分化形成多层肌管组织,采用自制体外细胞拉伸仪,对培养并分化的肌管进行间歇性应力加载:加载幅度10%,加载频率分别为0(A组)、0.25(B组)、0.50(C组)、1.00 Hz(D组),加载时间3次/d,每次1 h。连续加载5、7、10 d时,观察各组肌管形态;RT-PCR和实时荧光定量PCR(real-time fluorescent quantitative PCR,QRT-PCR)分析检测成肌相关基因成肌分化抗原(myogenic differentiationantigen,MyoD)、肌细胞生成素(Myogenin)、结蛋白(Desmin)、肌球重链蛋白(myosin heavy chain,MyHC)mRNA的表达差异。结果倒置相差显微镜观察示,应力加载促进各组肌管的极性融合及数量增加,其中B组加载培养7 d时,多层肌管排列紧密,极性显著。加载条件能促进成肌相关基因mRNA的表达:组内随加载时间延长,各组MyoD的mRNA表达逐渐下降,7、10 d与5 d比较差异均有统计学意义(P<0.05);Myogenin、Desmin、MyHC的mRNA表达呈先升高后降低趋势,以7 d表达量最高;B组除7 d与10 d Desmin的mRNA表达比较差异无统计学意义(P>0.05)外,其余各时间点比较差异均有统计学意义(P<0.05)。同时间点随加载频率增加,MyoD、Myogenin、Desmin、MyHC的mRNA表达呈先升高后降低趋势,以B组表达量最高;除5 d B、C组Desmin和10 d A、B组MyHC的mRNA表达比较差异无统计学意义(P>0.05)外,其余各组与B组各相关基因mRNA表达比较,差异均有统计学意义(P<0.05)。结论低频(0.25 Hz)、适时(7 d)的周期性应力加载有利于生长于Sylgard 184弹性材料表面的多层肌管极性分化,但随着应力加载时间延长,肌管的老化加速。

Effects of mechanical stretch with variant frequencies on alignment and differentiation of multilayer myotubes cultured in vitro

Objective To explore the e ects of mechanical stretch with variant frequencies on the alignment and di erentiation of the multilayer myotubes cultured in vitro,and to select the optimized cultural condition of regenerative skeletal muscle tissue with stress loading cultured in vitro.Methods C2C12 myoblasts cultured in vitro in the groove casts of Sylgard 184 were induced into the multilayer myotubes.Meanwhile the myoblasts were treated with various mechanical stretch with cells tensile instrument,at the amplitude of 10% and the frequency of 0(group A),0.25(group B),0.50(group C),and 1.00 Hz(group D) for 1 hour,3 times a day.The myotubes morphology was observed by inverted phase contrast microscope at 5,7,and 10 days after continuous mechanical stretch.And the expressions of mRNA for myogenic di erentiation antigen(MyoD),Myogenin,Desmin,and myosin heavy chain(MyHC) were detected by RT-PCR and real-time uorescent quantitative PCR(QRT-PCR),respectively.Results The mechanical stretch could promote the aligned fusion and increase the number of myotubes.Indeed the multilayer myotubes arranged more closely in group B at 7 days.At the same group,as the time went on,the mRNA expressions of MyoD gradually declined in each group.There were signi cant di erences in mRNA expressions of MyoD between 5 days and 7,10 days(P < 0.05).The mRNA expressions of Myogenin,Desmin,and MyHC were highest at 7 days.There were signi cant di erences between di erent time points(P < 0.05),except the mRNA expression of Desmin of group B between 7 and 10 days(P > 0.05).At the same time,with the increase of frequency,the highest mRNA expressions of MyoD,Myogenin,Desmin,and MyHC were in group B.There were signi cant di erences at the same time between group B and the other groups(P < 0.05),except mRNA expression of Desmin at 5 days between groups B and C,and mRNA expression of MyHC at 10 days between groups A and B(P > 0.05).Conclusion Low frequency(0.25 Hz) and suitable time(7 days) periodic mechanical stretch is bene cial to the di erentiation of the multilayer myotubes cultured in the groove casts of Sylgard 184,but as the stretch time goes on the aging of myotubes will be accelerated.