miRNA-206及其上游转录因子MyoD在大鼠骨骼肌损伤再修复过程中的表达

发布时间：2018-06-07 03:37

本文选题：miR-206 + MyoD　；参考：《上海体育学院》2014年硕士论文

【摘要】：研究目的运动性骨骼肌损伤和再修复是一个复杂连续的病理过程，其中涉及到许多生长因子的调节。在损伤阶段有肌纤维坏死和炎症因子的浸润，修复阶段有骨骼肌卫星细胞的激活增殖分化。近年来，已有研究证实有许多miRNAs参与了骨骼肌各项生命生理活动的调节，其中miR-206是骨骼肌中特有的一种miRNA。本研究旨在通过Armstrong建立的动物EIMI模型，采用组织学的方法观察一次性下坡跑后大鼠腓肠肌的肌纤维形态学方面的变化，并对骨骼肌特异性miR-206及其上游转录因子MyoD在损伤修复阶段的表达量的变化进行观察。进一步完善和丰富骨骼肌损伤再修复过程的分子机制，为临床运动损伤和肌肉病变提供新的理论依据和治疗手段。研究方法 72只2月龄运动能力良好的雄性SD大鼠，随机分为运动组和对照组。运动组分为运动后0h组、运动后6h组、运动后12h组、运动后24h组、运动后48h组、运动后72h组、运动后1w组、运动后2w组，每组8只。正式进行运动干预时期，对照组不进行干预，其它运动组的大鼠都进行一次持续性下坡跑的运动干预，速度为16m/min，坡度为-16°，总运动时间90min。运动结束后，分别在0h、6h、12h、1d、2d、3d、1w、2w后进行麻醉动物，迅速取出双侧腓肠肌。进行以下实验： 1）采用HE染色进行组织学鉴定，观察骨骼肌在各个时期形态结构的变化； 2）采用western blot方法检测TGF-β1、MyoD蛋白的表达情况； 3）采用RT-PCR实验对TGF-β1、MyoD mRNA水平以及骨骼肌特异性miR-206表达变化进行检测。研究结果 1）组织学HE染色：C组为形态结构正常的骨骼肌细胞，运动后6h发现骨骼肌肌纤维有圆形的，提示有肿胀的发生，24h后肿胀仍在存在且逐渐加重，48h后发现有炎症细胞浸润，72h嗜碱性颗粒最多，提示损伤程度最为严重，1w后偶见炎症细胞，肌纤维无肿胀，2w观察到肌纤维基本恢复到正常形态结构。 2）Western blot实验：一次性下坡跑后，大鼠腓肠肌TGF-β1蛋白表达水平除1w组外，其它各组较C组明显下降，0-48h表达逐渐下降，随后开始上升，2w时恢复至接近正常水平。而在运动后48h、1w、2w MyoD蛋白表达较C组有所上升；运动后0-48h的表达逐渐增加，48h达到峰值，72h表达下降，，1w时表达又上升。 3）RT-PCR实验：一次性下坡跑后，MyoD mRNA在48h、1w、2w组的表达较C组稍有上升； TGF-β1mRNA在运动后的表达较C组都有上升，而miR-206的表达较对照组整体下降；TGF-β1mRNA从运动后6h到24h表达逐渐增加，之后开始下降； MyoD mRNA和miR-206的表达均在运动6-12h开始随时间逐渐增加，12h达到峰值，之后开始下降，2w后下降到正常水平。结论 1）一次性下坡跑运动可导致大鼠腓肠肌损伤，损伤后2w基本恢复到正常水平。 2)在一次下坡跑过程中，MyoD可能激活了miR-206的表达，且促进了骨骼肌损伤的修复。
[Abstract]:Research purpose Exercise-induced skeletal muscle injury and rerepair is a complicated and continuous pathological process involving the regulation of many growth factors. There were myofibrotic necrosis and inflammatory factor infiltration in injury stage, and activation, proliferation and differentiation of skeletal muscle satellite cells in repair stage. In recent years, many studies have confirmed that there are many miRNAs involved in the regulation of various physiological activities of skeletal muscle, among which miR-206 is a unique type of miRNA in skeletal muscle. The purpose of this study was to observe the changes of muscle fiber morphology of gastrocnemius muscle in rats after once downhill running by means of EIMI model established by Armstrong. The expression of skeletal muscle specific miR-206 and its upstream transcription factor MyoD in the repair stage was observed. Further improve and enrich the molecular mechanism of skeletal muscle injury and rerepair process, and provide a new theoretical basis and treatment for clinical sports injury and muscle disease. Research method 72 male Sprague-Dawley rats were randomly divided into exercise group and control group. The exercise group was divided into 3 groups: 0 h after exercise, 6 h after exercise, 12 h after exercise, 24 h after exercise, 48 h after exercise, 72 h after exercise, 1 week after exercise and 2 weeks after exercise with 8 rats in each group. During the period of formal exercise intervention, the control group did not intervene. The rats in the other exercise group were given a continuous downhill running exercise intervention with a speed of 16 m / min, a slope of -16 掳and a total exercise time of 90 min. At the end of exercise, the anesthetized animals were taken out of the bilateral gastrocnemius muscle at 0 h, 6 h, 12 h, 1 day, 2 d, 3 d and 1 week after 2 weeks of anaesthesia, and the bilateral gastrocnemius muscle was removed quickly. The following experiments were carried out: 1) histopathological identification was carried out by HE staining to observe the morphological changes of skeletal muscle in different periods. 2) the expression of TGF- 尾 _ 1 and MyoD protein was detected by western blot method. 3) the level of TGF- 尾 _ 1 MyoD mRNA and the expression of skeletal muscle specific miR-206 were detected by RT-PCR assay. Research results 1) the skeletal muscle cells in group 1: C were found to have round muscle fibers at 6 h after exercise. The results indicated that the swelling still existed 24 hours after the occurrence of swelling and gradually increased after 48 hours. The most alkalophilic granules were found after 72 hours of infiltration of inflammatory cells, suggesting that inflammatory cells were occasionally found after 1 week of most severe injury. No swelling of muscle fiber was observed for 2 weeks. 2)Western blot test: the expression level of TGF- 尾 1 protein in gastrocnemius muscle of rats was significantly decreased after downhill running at one time, except for 1 week group, and gradually decreased from 0 to 48 hours in other groups than that in group C, and then returned to the normal level at 2w. However, the expression of MyoD protein increased at 1 week after exercise and increased gradually from 0 to 48 hours after exercise, and then increased at the end of 72 hours after exercise, and then increased at 1 week after exercise. 3)RT-PCR experiment: the expression of MyoD mRNA in group C was slightly higher than that in group C after one downhill run at 48h and 1ww, and the expression of TGF- 尾 1mRNA was higher than that of group C after exercise, while the expression of miR-206 decreased gradually from 6h to 24h after exercise, and the expression of TGF- 尾 1mRNA increased gradually compared with control group from 6 h to 24 h after exercise. Then the expression of; MyoD mRNA and miR-206 began to decrease at 6-12 h after exercise and gradually increased to a peak at 12 h, then decreased to normal level after 2 weeks. Conclusion 1) once downhill running can cause gastrocnemius muscle injury in rats, and return to normal level 2 weeks after injury. 2) during a downhill run, MyoD may activate the expression of miR-206 and promote the repair of skeletal muscle injury.
【学位授予单位】：上海体育学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：G804.2

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