本文選題：miR-206 + MyoD��； 參考：《上海體育學院》2014年碩士論文

【摘要】：研究目的 運動性骨骼肌損傷和再修復是一個復雜連續(xù)的病理過程，其中涉及到許多生長因子的調(diào)節(jié)。在損傷階段有肌纖維壞死和炎癥因子的浸潤，修復階段有骨骼肌衛(wèi)星細胞的激活增殖分化。近年來，已有研究證實有許多miRNAs參與了骨骼肌各項生命生理活動的調(diào)節(jié)，其中miR-206是骨骼肌中特有的一種miRNA。本研究旨在通過Armstrong建立的動物EIMI模型，采用組織學的方法觀察一次性下坡跑后大鼠腓腸肌的肌纖維形態(tài)學方面的變化，并對骨骼肌特異性miR-206及其上游轉錄因子MyoD在損傷修復階段的表達量的變化進行觀察。進一步完善和豐富骨骼肌損傷再修復過程的分子機制，為臨床運動損傷和肌肉病變提供新的理論依據(jù)和治療手段。 研究方法 72只2月齡運動能力良好的雄性SD大鼠，隨機分為運動組和對照組。運動組分為運動后0h組、運動后6h組、運動后12h組、運動后24h組、運動后48h組、運動后72h組、運動后1w組、運動后2w組，每組8只。正式進行運動干預時期，對照組不進行干預，其它運動組的大鼠都進行一次持續(xù)性下坡跑的運動干預，速度為16m/min，坡度為-16°，總運動時間90min。運動結束后，分別在0h、6h、12h、1d、2d、3d、1w、2w后進行麻醉動物，迅速取出雙側腓腸肌。 進行以下實驗： 1）采用HE染色進行組織學鑒定，觀察骨骼肌在各個時期形態(tài)結構的變化； 2）采用western blot方法檢測TGF-β1、MyoD蛋白的表達情況； 3）采用RT-PCR實驗對TGF-β1、MyoD mRNA水平以及骨骼肌特異性miR-206表達變化進行檢測。 研究結果 1）組織學HE染色：C組為形態(tài)結構正常的骨骼肌細胞，運動后6h發(fā)現(xiàn)骨骼肌肌纖維有圓形的，提示有腫脹的發(fā)生，24h后腫脹仍在存在且逐漸加重，48h后發(fā)現(xiàn)有炎癥細胞浸潤，72h嗜堿性顆粒最多，提示損傷程度最為嚴重，1w后偶見炎癥細胞，肌纖維無腫脹，2w觀察到肌纖維基本恢復到正常形態(tài)結構。 2）Western blot實驗：一次性下坡跑后，大鼠腓腸肌TGF-β1蛋白表達水平除1w組外，其它各組較C組明顯下降，0-48h表達逐漸下降，隨后開始上升，2w時恢復至接近正常水平。而在運動后48h、1w、2w MyoD蛋白表達較C組有所上升；運動后0-48h的表達逐漸增加，48h達到峰值，72h表達下降，，1w時表達又上升。 3）RT-PCR實驗：一次性下坡跑后，MyoD mRNA在48h、1w、2w組的表達較C組稍有上升； TGF-β1mRNA在運動后的表達較C組都有上升，而miR-206的表達較對照組整體下降；TGF-β1mRNA從運動后6h到24h表達逐漸增加，之后開始下降； MyoD mRNA和miR-206的表達均在運動6-12h開始隨時間逐漸增加，12h達到峰值，之后開始下降，2w后下降到正常水平。 結論 1）一次性下坡跑運動可導致大鼠腓腸肌損傷，損傷后2w基本恢復到正常水平。 2)在一次下坡跑過程中，MyoD可能激活了miR-206的表達，且促進了骨骼肌損傷的修復。
[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 擄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.
【學位授予單位】：上海體育學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：G804.2

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