盆底骨骼肌细胞机械刺激后肌纤维的改变

Changes in muscle fibers of pelvic floor skeletal muscle cells following mechanical stimulation

目的研究盆底骨骼肌细胞在受到机械力刺激情况下Ⅰ型及Ⅱ型肌纤维含量的改变及肌球蛋白重链亚型(MHCⅠ、MHCⅡa、MHCⅡb和MHCⅡx)的改变。方法选取2012年3月行直肠癌根治术患者的肛提肌组织,显微镜下大体组织标本鉴定为肛提肌组织,且无肿瘤浸润。采用两步消化法及差速贴壁法进行细胞培养;采用Flexcell-4000细胞柔性基底拉伸系统进行对细胞进行正弦波的拉伸应变加载实验(频率为3Hz,加力强度分别为3%、6%),以未加载力的静态细胞作为对照组。用ATP酶染色法鉴定Ⅰ型和Ⅱ型肌纤维并比较对照组及受力6、24hⅠ型及Ⅱ型肌纤维含量的改变;用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)凝胶电泳观察肌球蛋白重链亚型在不同受力时间的改变。结果①加力强度为3%,加力时间为6h使Ⅰ型肌纤维的面密度增加(P<0.01),Ⅱ型肌纤维的面密度减少(P<0.05)。②加力强度为3%,加力时间为24h;加力强度为6%,加力时间为6h使Ⅰ型肌纤维的面密度减少,Ⅱ型肌纤维的面密度增加(P<0.05)。③加力强度为3%,加力时间为6h使肌纤维MHCⅠ%升高,MHCⅡb%降低(P<0.01);加力强度为3%,加力时间为24h;加力强度为6%,加力时间为6h使肌纤维MHCⅠ%和MHCⅡb%降低,MHCIIa%升高(P<0.01)。结论不同受力情况MHC亚型之间发生变化,加力强度3%加力时间6h机械力刺激引起MHCⅡb向MHCⅡx、MHCⅡa、MHCⅠ方向变化;加力强度3%加力时间24h、加力强度6%加力时间6h和加力强度6%加力时间24h机械力刺激引起MHCⅠ、MHCⅡb向MHCⅡx、MHCⅡa变化。

Objective To study the changes in type I and 11 muscle fibers and myosin heavy chain subtypes （MHC I, MHC I1 a, MHC I1 b and MHC I1 x） of the pelvic floor skeletal muscle cells following mechanical stretch stimulation. Methods We examined the levator ani tissues of patients received rectal cancer radical resection in March 2012. The levator ani tissue free of tumor infiltration was identified under microscope. A two-step digestion method was employed to conduct differential adherent cell culture. The Flexcell-4000 cell flexible substrate stretching system was applied to perform sine wave tension-strain loading experiments （frequency： 3 Hz, intensity： 3% and 6%）, in which the static cells without loading served as the control group. The type I and type II muscle fibers were identified by ATP enzyme staining, thus allowing comparison with the control group regarding the changes in muscle fibers following 6 and 24 hours of tension stretching. The SDS-PAGE gel electrophoresis was conducted to determine the changes in myosin heavy chain subtypes at different time points following tension stretching. Results The 6-hour ad- ministration of mechanical stretch at an intensity of 3% resulted in an increase in the surface density of type I muscle fibers and MHC 1% （both P〈0.01） as well as a reduction in the surface density of type II muscle fibers （P〈O.05） and MHC 11 b% （P〈0.01）. The 24-hour administration of mechanical stretch at an intensity of 3% followed by a 6-hour me- chanical stretch at an intensity of 6% led to a decrease in the surface density of type I muscle fibers （P〈0.05）, MHC 1% and MHC II b% （both P〈0.01） as well as an increase in type I1 muscle fibers （P〈0.05） and MHC I1 a% （P〈0.01）. Conclusion Different extents of mechanical stretches result in altered proportion of MHC subtypes. This is evidenced by the fact that 6-hour administration of mechanical stretch at an intensity of 3% resulted in a shift of MHC I1 b to MHC II x, MHC I1 a and MHC I and that 24-hour administration of mechanical stretch at an intensity of 3% followed by a 6- hour mechanical stretch at an intensity of 6% led to the shift of MHC I to MHC I1 x, MHC II a and MHC I1 b.