miR-98在运动性骨骼肌损伤修复过程中作用的研究

发布时间：2018-02-28 23:29

  本文关键词： miR-98 运动性骨骼肌损伤 E2F5 ID1　出处：《上海体育学院》2017年硕士论文　论文类型：学位论文

【摘要】：研究目的:随着人们观念的改变和大众健身的普及,运动性骨骼肌损伤已经不仅存在于竞技体育,也成为困扰大众健身的常见问题。损伤后的修复会出现一系列问题,如:瘢痕、纤维化、疼痛等。相关研究发现:miR-98可以调控骨骼肌卫星细胞的分化,使其成为骨骼肌损伤修复的又一靶点。本实验通过研究下坡跑大鼠股直肌损伤修复过程中miR-98的作用,为运动性骨骼肌损伤的治疗提供新的理论基础和实验依据。研究方法:72只两月龄健康雄性SD大鼠,随机分为9组,每组8只。分组:安静对照组(C)、运动后0 h组、运动后6 h组、运动后12h组、运动后24 h组、运动后48 h组、运动后72 h组、运动后1 W组、运动后2 W组。利用下坡跑建立运动性骨骼肌损伤实验模型,运动方案参数:跑台速度为16m/min,坡度为-16°,运动时长为120min。用10%的水合氯醛腹腔注射麻醉,通过下腔静脉取血,离心取血清后保存在-80℃C冰箱;取血后迅速取股直肌,肌肉标本迅速放入用液氮冷却的异戊烷中冷冻10s左右;完全冷冻后放于干冰上,待异戊烷完全挥发后,迅速用锡箔纸包上放入超低温冰箱储存。所取组织样本进行以下实验:1)股直肌进行冰冻切片HE染色(横切)进行形态学观察;2)采用比色法和ELISA试剂盒分别对血清中CK和CK-MM的变化进行检测;3)采用RT-PCR对股直肌中miR-98、E2F5、ID1 mRNA水平进行检测;4)采用Western-Blot实验对股直肌中E2F5、ID1蛋白表达情况进行检测。研究结果:1.HE染色结果:对照组结果显示:肌纤维成多边形,大小均一,排列紧密规则,肌细胞核均匀的分布在细胞膜下;运动后Oh、6h、12h、24h、48h、72h组切片显示:肌纤维排列散乱,细胞间隙变大,形状不规则,呈肿胀状态;运动后1w和2w组切片显示:肌纤维形态和对照组相似,趋于正常。2.血清 CK 和 CK-MM:与C组相比,运动后Oh组血清CK的含量明显升高,具有显著性差异(P0.05);运动后6h组血清CK含量几乎持平;运动后12h、24h、48h组血清CK含量稍有升高,无显著性差异;运动后72h、1W、2W组血清CK含量稍微降低,但无显著性差异。运动后大鼠血清CK-MMELISA结果显示:与C组相比,运动后Oh、6h、12h组血清中CK-MM的含量增加,无显著性差异;运动后24h血清中CK-MM的含量显著升高,具有显著性差异(P0.05);运动后48h和72h血清中含量上升,无显著性差异;运动后1W和2W后,血清CK-MM的含量已经基本恢复。3.运动后股直肌中miR-98、E2F5和ID1 mRNA水平变化:与C组相比,运动后Oh和6h组miR-98 mRNA的含量稍有升高,无显著性差异;运动后12h组miR-98 mRNA含量下降,无显著性差异;运动后24h、48h和72h组miR-98 mRNA含量明显下降,具有显著性差异(P0.05);运动后1W组miR-98 mRNA含量稍降低,无显著性差异;运动后W组miR-98 mRNA含量稍升高,无显著性差异。与C组相比,运动后Oh组E2F5mRNA含量升高,无显著性差异;运动后6h、12h和24h组E2F5 mRNA含量明显升高,具有显著性差异(P0.05);运动后48h和72h组E2F5 mRNA含量上升,无显著性差异;运动后1W和2W组E2F5 mRNA含量稍有降低,无显著性差异。与C组ID1 mRNA含量相比,运动后Oh和6h组ID1 mRNA含量增加,无显著性差异;运动后12h组ID1 mRNA含量降低,无显著性差异;运动后24h、48h和72h组ID1 mRNA含量明显下降,具有显著性差异(P0.05);运动后1W和2W组ID1 mRNA含量降低,但无显著性差异。4.运动后各组股直肌中E2F5、ID1蛋白含量的变化:与C组相比,运动后0h、6h、12h、24h和48h组E2F5蛋白含量明显升高,具有显著性差异(P0.05);运动后72h、1W和2W组E2F5蛋白含量稍有升高,但无显著性差异。与C组相比,运动后0h、6h和12h组股直肌中ID1含量下降,但无显著性差异;运动后24h和48h组股直肌中ID1含量明显下降,具有显著性差异(P0.05);运动后72h、1W和2W组股直肌中ID1含量降低,但无显著性差异。研究结论:1.一次长时间离心运动可以成功建立运动性骨骼肌损伤实验模型;2.运动性骨骼肌损伤时miR-98含量下降,促进骨骼肌损伤的修复。
[Abstract]:Objective: with the popularity of the people idea change and the public fitness, exercise induced skeletal muscle injury has not only exist in the sports, has become a common problem of public health. The repair of the injured will appear a series of problems, such as scarring, fibrosis, pain and so on. The study found that miR-98 can regulate differentiation of skeletal muscle satellite cells, which has become a target of skeletal muscle injury. In this experiment, miR-98 ran downhill rectus femoris of rats during the injury and repair effect through research, to provide theoretical and experimental basis for the new treatment of exercise-induced skeletal muscle injury. Methods: 72 of 2 month old healthy male SD rats were randomly divided into 9 groups, 8 rats in each group. Group: normal control group (C), 0 h after exercise group, 6 h after exercise group, 12h after exercise group, exercise group after 24 h, 48 h after exercise group, exercise group after 72 h, 1 W after exercise group, W after exercise 2 group. By running the establishment of exercise-induced skeletal muscle injury experimental model of downhill exercise program parameters: treadmill speed of 16m/min, the slope is -16 degrees, the movement for a long time by intraperitoneal injection with 10% chloral hydrate 120min. anesthesia, the inferior vena cava blood, serum were stored in the -80 C C refrigerator; blood samples taken immediately after the rectus femoris muscle specimens, quickly put frozen in isopentane cooled in liquid nitrogen is about 10s; after completely frozen on dry ice, to isopentane completely volatilized quickly using the foil bag into the ultra low temperature freezer storage. The tissue samples were the following experiments: 1) of rectus femoris HE staining (cross) were observed; 2) by colorimetric method and ELISA kit respectively on the change of CK and CK-MM in serum were detected by RT-PCR E2F5; 3) of miR-98, ID1 were detected in the rectus femoris, mRNA level; 4) using Western-Blot experiments of rectus femoris E2F5, ID1 protein expression were detected. Results: the results of 1.HE staining: the control group showed that muscle fiber into polygon, uniform size, closely arranged rules, muscle cells evenly distributed in the cell membrane; Oh after exercise, 6h, 12h, 24h, 48h, 72h group section showed that the muscle fibers were scattered and the cell gap becomes larger, irregular shape, showed swelling of the state; and 1W after exercise group 2W section showed that muscle fiber morphology and similar to the control group, serum CK and CK-MM: became normal in.2. compared with C group, the content of serum CK in Oh group increased significantly after exercise, with a significant difference (P0.05); after exercise 6h serum CK was almost unchanged after exercise; 12h, 24h, 48h group, serum CK content increased slightly, no significant difference; after exercise 72h, 1W, 2W group of serum CK content decreased slightly, but there was no significant difference. The results show CK-MMELISA in the serum of rats after exercise: compared with C group, after exercise Oh, 6h, 1 Increase the content of CK-MM in serum of 2H group, no significant difference; the content of CK-MM 24h in serum after exercise was significantly increased, with significant difference (P0.05); after exercise the content of 48h and 72h in serum increased, no significant difference; after exercise 1W and 2W, the content of serum CK-MM has returned to.3. after the rectus femoris miR-98, E2F5 and ID1 change mRNA level: compared with C group, the content of Oh and 6h after exercise group miR-98 mRNA increased slightly, no significant difference; after exercise group 12h miR-98 mRNA decreased, no significant difference; after exercise 24h, 48h and 72h group miR-98 mRNA was significantly decreased, with significant difference (P0.05); 1W after exercise group miR-98 mRNA content decreased slightly, no significant difference; W group miR-98 mRNA content increased slightly after exercise, there was no significant difference. Compared with the C group, Oh group increased the content of E2F5mRNA after exercise, there was no significant difference; after exercise 6h, 12h and 24h group E2F5 mRNA鍚�閲忔槑鏄惧崌楂�,鍏锋湁鏄捐憲鎬у樊寮，

本文编号：1549461