本文選題：miR-206 + MyoD�。� 參考：《上海體育學(xué)院》2014年碩士論文

【摘要】：研究目的 運(yùn)動(dòng)性骨骼肌損傷和再修復(fù)是一個(gè)復(fù)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(fù)階段有骨骼肌衛(wèi)星細(xì)胞的激活增殖分化。近年來，已有研究證實(shí)有許多miRNAs參與了骨骼肌各項(xiàng)生命生理活動(dòng)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(dòng)物EIMI模型，采用組織學(xué)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(xué)方面的變化，并對(duì)骨骼肌特異性miR-206及其上游轉(zhuǎn)錄因子MyoD在損傷修復(fù)階段的表達(dá)量的變化進(jìn)行觀察。進(jìn)一步完善和豐富骨骼肌損傷再修復(fù)過程的分子機(jī)制，為臨床運(yùn)動(dòng)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(yùn)動(dòng)能力良好的雄性SD大鼠，隨機(jī)分為運(yùn)動(dòng)組和對(duì)照組。運(yùn)動(dòng)組分為運(yùn)動(dòng)后0h組、運(yùn)動(dòng)后6h組、運(yùn)動(dòng)后12h組、運(yùn)動(dòng)后24h組、運(yùn)動(dòng)后48h組、運(yùn)動(dòng)后72h組、運(yùn)動(dòng)后1w組、運(yùn)動(dòng)后2w組，每組8只。正式進(jìn)行運(yùn)動(dòng)干預(yù)時(shí)期，對(duì)照組不進(jìn)行干預(yù)，其它運(yùn)動(dòng)組的大鼠都進(jìn)行一次持續(xù)性下坡跑的運(yùn)動(dòng)干預(yù)，速度為16m/min，坡度為-16°，總運(yùn)動(dòng)時(shí)間90min。運(yùn)動(dòng)結(jié)束后，分別在0h、6h、12h、1d、2d、3d、1w、2w后進(jìn)行麻醉動(dòng)物，迅速取出雙側(cè)腓腸肌。 進(jìn)行以下實(shí)驗(yàn)： 1）采用HE染色進(jìn)行組織學(xué)鑒定，觀察骨骼肌在各個(gè)時(shí)期形態(tài)結(jié)構(gòu)的變化； 2）采用western blot方法檢測TGF-β1、MyoD蛋白的表達(dá)情況； 3）采用RT-PCR實(shí)驗(yàn)對(duì)TGF-β1、MyoD mRNA水平以及骨骼肌特異性miR-206表達(dá)變化進(jìn)行檢測。 研究結(jié)果 1）組織學(xué)HE染色：C組為形態(tài)結(jié)構(gòu)正常的骨骼肌細(xì)胞，運(yùn)動(dòng)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(xì)胞浸潤，72h嗜堿性顆粒最多，提示損傷程度最為嚴(yán)重，1w后偶見炎癥細(xì)胞，肌纖維無腫脹，2w觀察到肌纖維基本恢復(fù)到正常形態(tài)結(jié)構(gòu)。 2）Western blot實(shí)驗(yàn)：一次性下坡跑后，大鼠腓腸肌TGF-β1蛋白表達(dá)水平除1w組外，其它各組較C組明顯下降，0-48h表達(dá)逐漸下降，隨后開始上升，2w時(shí)恢復(fù)至接近正常水平。而在運(yùn)動(dòng)后48h、1w、2w MyoD蛋白表達(dá)較C組有所上升；運(yùn)動(dòng)后0-48h的表達(dá)逐漸增加，48h達(dá)到峰值，72h表達(dá)下降，，1w時(shí)表達(dá)又上升。 3）RT-PCR實(shí)驗(yàn)：一次性下坡跑后，MyoD mRNA在48h、1w、2w組的表達(dá)較C組稍有上升； TGF-β1mRNA在運(yùn)動(dòng)后的表達(dá)較C組都有上升，而miR-206的表達(dá)較對(duì)照組整體下降；TGF-β1mRNA從運(yùn)動(dòng)后6h到24h表達(dá)逐漸增加，之后開始下降； MyoD mRNA和miR-206的表達(dá)均在運(yùn)動(dòng)6-12h開始隨時(shí)間逐漸增加，12h達(dá)到峰值，之后開始下降，2w后下降到正常水平。 結(jié)論 1）一次性下坡跑運(yùn)動(dòng)可導(dǎo)致大鼠腓腸肌損傷，損傷后2w基本恢復(fù)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(dá)，且促進(jìn)了骨骼肌損傷的修復(fù)。
[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(xué)位授予單位】：上海體育學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：G804.2

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