被动运动干预对大鼠失神经萎缩骨骼肌中miRNA-1表达和成肌细胞分化的影响

EFFECT OF PASSIVE MOVEMENT ON EXPRESSION OF miRNA-1 AND DIFFERENTIATION OF MYOBLASTS IN DENERVATION-INDUCED SKELETAL MUSCLE ATROPHY IN RATS

目的探讨mi RNA-1在大鼠早期失神经支配后骨骼肌中不同时段的表达,以及被动运动对失神经骨骼肌mi RNA-1表达及成肌细胞分化的影响。方法取SD大鼠27只,体质量(200±10)g,随机分为假手术组(A组,n=3)、失神经组(B组,n=12)和失神经被动运动组(C组,n=12)。各组大鼠显露右侧坐骨神经并游离,B、C组切除坐骨神经长约1 cm,A组仅显露游离神经不切除;术后1 d起,对C组大鼠右后肢行被动屈伸运动。A组于术后6 h,B、C组分别于术后3、7、14、28 d,采用颈椎脱臼法各处死3只大鼠,测量腓肠肌湿重比以及行HE染色观察腓肠肌细胞直径,对肌萎缩情况进行综合评价;采用RT-PCR法检测mi RNA-1、肌分化因子(myocyte differentiation factor,Myo D)基因表达,免疫组织化学染色观察Myo D蛋白表达。结果 B、C组腓肠肌失神经后均有不同程度萎缩,随失神经时间延长,肌纤维排列逐渐紊乱,肌细胞直径逐渐减小,失去正常的结构形态。B、C组术后各时间点腓肠肌湿重及肌细胞直径均小于A组(P<0.05);B、C组间除术后3 d腓肠肌湿重比以及术后3、28 d肌细胞直径比较差异无统计学意义(P>0.05)外,其余各时间点C组均大于B组(P<0.05)。术后随时间延长,B、C组mi RNA-1和Myo D基因相对表达量呈逐渐升高趋势,但各时间点均显著低于A组(P<0.05);B、C组间除术后3 d比较差异无统计学意义外(P>0.05),术后7、14、28 d C组mi RNA-1和Myo D基因相对表达量均高于B组(P<0.05)。免疫组织化学染色示A、B、C组各时间点Myo D均呈阳性表达,A组阳性表达少于B、C组;B、C组随失神经时间延长,阳性表达逐渐增多,且C组各时间点阳性表达均多于B组。相关性分析结果示mi RNA-1、Myo D分别与腓肠肌湿重比在3、7 d时无相关性(P>0.05),14、28 d时成正相关(P<0.05);各时间点Myo D与mi RNA-1基因相对表达量成正相关(P<0.05)。结论被动运动可能通过促进mi RNA-1的表达,以促进肌细胞分化发挥防止失神经骨骼肌萎缩的作用。

Objective To investigate the expression of miRNA- 1 in denervated skeletal muscle at different periods, and to explore effects of passive movement on the expression of miRNA-1 and differentiation of myoblasts in denervation-induced skeletal muscle atrophy in rats. Methods Twenty-seven Sprague Dawley rats, weighing （200_＋10） g, were randomly divided into sham-operated group （group A, n=3）, denervated group （group B, n=12）, and passive movement group （group C, n=12）. After the right sciatic nerve was exposed and dissociated, the sciatic nerve of 1 cm in length was removed in groups B and C; resection was not performed in group A. At 1 day after operation, passive flexion and extension movement was performed on the right hind limb in group C. At 6 hours in group A and at 3, 7, 14, and 28 days in groups B and C, 3 rats were sacrificed to measure the wet weight ratio of gastrocnemius muscle, to observe the diameter of the gastrocnemius muscle cell and evaluate the muscle atrophy by HE staining; RT-PCR was used to detect the mRNA expression of miRNA-1 and myocyte differentiation factor （MyoD）, and immunohistochemistry to determine the protein expression of MyoD. Results Atrophy in various degrees was observed in denervated gastrocnemius muscle of groups B and C. The muscle fiber arranged in disorder and the diameter of the muscle cells decreased gradually with the time, without normal structure and morphology. The wet weight ratio and the cell diameter of the gastrocnemius in groups B and C were significantly less than those in group A （P〈0.05）; the wet weight ratio at 7, 14, 28 days and the cell diameter at 7, 14 days of group B were significantly greater than those of group A （P〈O.05）. The expressions of miRNA-1 and MyoD mRNA gradually increased with time in groups B and C, but were significantly less than those of group A at each time point （P〈O.05）. At 7, 14, and 28 days after operation, the expressions of miRNA-1 and MyoD mRNA in group C were significantly higher than those in group B （P〈O.05）. Immunohistochemical staining showed positive expression of MyoD in groups A, B, and C at each time point, but higher expression was observed in groups B and C than group A; the expression increased with time in groups B and C, and it was significantly higher in group C than group B. The correlation analysis results showed that the overall change trend of miRNA-1 and MyoD had no relation with the gastrocnemius wet weight ratio at 3 and 7 days （P〉O.05）, and had positive correlation at 14 and 28 days （P〈0.05）; positive correlation was found between the relative expression of MyoD and miRNA-1 mRNA （P〈O.05）. Conclusion Passive movement can prevent amyotrophy by increasing the expression of miRNA- 1 and promoting the differentiation of myoblasts.