经皮电刺激作用于失神经骨骼肌萎缩分子机制的研究

The molecule mechanisms of electric stimulus effects on the denervation muscle atrophy

目的探讨电刺激作用于失神经骨骼肌萎缩的分子机制。方法选用Wista大鼠54只,随机分为A,B,C,D,E,F,G,H,I 9组,每组6只。48只大鼠制作成标准的右侧坐骨神经离断、腓肠肌失神经支配模型,A组为对照组(假手术组),B,C,D,E组为去神经组,F,G,H,I组为电刺激组。术后2、7、14、28 d选取腓肠肌样本,应用反转录-聚合酶链反应(RT-PCR)检测不同时段的MuRF1和MyoD基因转录,应用和West blotting检测MuRF1不同时段的蛋白质表达水平,各组均以GAPDH为内参,应用免疫组织化学检测MyoD不同时段的蛋白质表达变化。结果去神经组与对照组比较,2、7、14、28 d MuRF1的mRNA持续增加(P<0.05),但2 d组增加不明显(P>0.05)。蛋白质水平先降低(7 d最低),随后增加(P<0.05)。干预组和去神经组比较,mRNA和蛋白表达水平均降低(P<0.05)。去神经组和干预组与对照组对比,MyoD的mRNA和蛋白持续增加(P<0.05),去神经组和干预组对比,mRNA和蛋白持续减少(P<0.05)。结论电刺激通过抑制蛋白酶体途径和增加成肌因子,起到延缓失神经骨骼肌萎缩的作用。

Objective To investigate the molecule mechanisms of the effect of electric stimulus on denervation muscle atrophy. Methods Fifty-four male Wista rats were randomly divided into nine groups and six rats were in each group. Standard model of denervated gastrocnemius muscle was established in right lower limb of group B to I. group A： control group（Sham operation were made only for the rats in control group）; group B to E： denervated group; group F to I： denervated and electric stimulus. Gastrocnemius muscle specimens were obtained at 2,7,14 and 28 days after operation. Total RNA extraction and RT-PCR procedures were done in-gastroenemius muscle specimens at different time following procedures were done in gastrocnemius muscle specimens at different time following operation. Forwards and reversre primers of MuRF1 and Myod for PCR were designed using Premier software and RT-PCR method, using GAPDH as an internal standard, was applied to study the expression of specific mRNAs for MuRF1 and Myod. Semiquantitative protein expression results of MuRF1 were obtained using West blotting method taking GAPDH as an interhal standard and MyoD protein level by immunohistochemistry to make the statisticial analyasis. Results denervated group compared to control group, the mRNA of MuRF1 increased instantly except group 2 d, but there protein decreased at early stage（7 d） and up-regulated thereafter. Intervated group compared to denervation group, all of mRNA and protein of MuRF1 down-regulated, the expression of MyoD mRNA and MyoD protein level in specimens of denervated group and denervated and electric stimulus group are up-regulated at 2 d,7 d,14 d,28 d after denervation compared with that in Group A. Conclusion Murfl expression of gastrocnemius denervation model of rats can be down-regulated but up-regulated at the expression of MyoD by electric stimulus, which shows that electric stimulus is a effective way of delaying denervated skeletal muscle atrophy.