一次性下坡跑对大鼠股直肌内源性因子MyoD和Myogenin表达变化的影响

发布时间：2018-06-22 03:58

  本文选题：股直肌 + MyoD　； 参考：《上海体育学院》2016年硕士论文

【摘要】：研究目的通过对大鼠进行一次性下坡跑训练,建立运动性骨骼肌损伤动物模型。结合股直肌的组织形态学变化,同时检测血清CK和CK-MM的变化,探究在运动性骨骼肌损伤修复过程中Myo D和Myogenin的表达变化情况。研究方法选取72只两月龄健康雄性SD大鼠为实验对象,将其随机分为9组,每组8只。分组情况如下:a.安静对照组(C);b.运动后即刻组(T0h);c.运动后6小时组(T6h);d.运动后12小时组(T12h);e.运动后24小时组(T24h);f.运动后48小时组(T48h);g.运动后72小时组(T72h);h.运动后1周组(T1w);i.运动后2周组(T2w)。结合Armstrong离心运动诱导运动性骨骼肌损伤实验模型以及课题组之前的运动实验方案,确定运动组的运动方案。运动方案具体参数如下:跑台速度为16m/min,坡度为-16°,运动时长为120min。对照组不进行下坡跑运动直接取材,运动训练各组分别在运动后0小时、6小时、12小时、24小时、48小时、72小时、1周、2周进行取材。所有实验对象均使用10%的水合氯醛腹腔注射进行麻醉。通过腹主动脉取血,离心取血清后保存在超低温冰箱待用;此外,迅速分离出股直肌,所取肌肉标本利用液氮冷却的异戊烷进行快速冷冻,之后置于干冰上待异戊烷挥发完全后于超低温冰箱保存备用。所取组织样本进行以下各项实验:1)采用股直肌冰冻切片HE染色(横切)进行组织形态学分析;2)采用比色法和ELISA试剂盒分别对血清CK和CK-MM水平进行检测;3)采用RT-PCR实验对Myo D、Myogenin m RNA水平进行检测;4)采用Western-Blot实验对Myo D、Myogenin蛋白表达情况进行检测。研究结果1.HE染色结果:对照组组织切片染色结果显示肌纤维成多边形,大小均匀,排列紧密规则,肌细胞核可见于肌膜边缘;运动后不同时间组取材的组织切片结果与对照组的结果相比显示,肌纤维排列不紧密,间隙变大,形状不规则,呈肿胀状态。运动后6h组股直肌横切片就发现有肿胀的肌纤维,运动后12h组到运动后48h组肌纤维肿胀持续加重,并出现核集聚现象;运动后48组和运动后72h组的切片显示肌纤维排列最为散乱,间隙最大,核外溢最多。运动后1w组和运动后2w组的切片显示肌纤维分布趋于正常状态,肌纤维紧密排列。2.血清CK和CK-MM:血清CK结果显示该指标在运动后0h组、运动后6h组、运动后72h组及运动后1w组与对照组相比有统计学显著性差异。并且该指标在运动后0h组到运动后48h组表达水平在不断增高。血清CK-MM的结果显示该指标在运动后24h组、运动后48h组与对照组相比具有统计学显著性差异。并且该指标在运动后0h组到运动后24h组表达水平在不断增高,之后呈下降趋势。3.Myo D、Myogenin m RNA的表达变化:RT-PCR实验结果显示,Myo D m RNA表达水平在运动后12h组、运动后48h组、运动后72h组以及运动后1w组与对照组相比有统计学显著性差异(p0.05);Myogenin m RNA表达水平在运动后12h组、运动后24h组及运动后48h组与对照组相比有统计学显著性差异(p0.05);Id1 m RNA表达水平在运动后6h组和运动后2w组与对照组相比有统计学显著性差异(p0.05)。4.Myo D、Myogenin蛋白的表达变化:一次性下坡跑运动后,Myogenin蛋白的Western blot结果显示,运动后6h组、运动后12h组和对照组相比有统计学显著性差异(p0.05),同样是在运动后即刻开始下降,之后在运动后24h组达到最大值,后继续下降至运动前水平。一次性下坡跑运动后,Myo D蛋白的Western blot结果显示,运动后0h组、运动后6h组、运动后12h组和运动后1w组与对照组有统计学显著性差异(p0.05),并且在运动后即刻呈下降的趋势,在运动后24h组达到峰值后又继续下降至对照组水平。研究结论1.一次性长时间下坡跑运动可以成功诱导运动性骨骼肌损伤实验模型;2.结合血清CK和CK-MM的变化特点,血清CK可以用作运动性骨骼肌损伤评价的敏感指标,血清CK-MM作为辅助评价运动性骨骼肌损伤更准确;3.在一次性下坡跑诱导的运动性骨骼肌损伤中Myo D和Myogenin在转录水平存在时相性差异。
[Abstract]:The purpose of this study was to establish an animal model of athletic skeletal muscle injury by one-off downhill running training in rats. In combination with the histomorphological changes of the rectus femoris, the changes of serum CK and CK-MM were detected at the same time, and the changes of Myo D and Myogenin during the repair of sports skeletal muscle injury were investigated. The study method selected 72 2 month old. Healthy male SD rats were randomly divided into 9 groups, each group was divided into 8 rats in each group as follows: A. quiet control group (C), immediate group after exercise (T0h), 6 hour group after C. exercise (T6h), 12 hours after D. exercise (T12h), 24 hours after E. exercise (T24h), 48 hours after F. exercise, 72 hours after exercise; 1 weeks post exercise group 1W); 2 weeks after I. movement (T2w). Combined with the experimental model of motor skeletal muscle injury induced by Armstrong centrifugation and the program before the task group, the motion scheme was determined. The specific parameters of the exercise program are as follows: the speed of the runway is 16m/min, the slope is -16 degrees, and the long movement time is that the 120min. control group does not carry out the downhill running straight. All the groups were taken 0 hours, 6 hours, 12 hours, 24 hours, 48 hours, 48 hours, 72 hours, 1 weeks and 2 weeks after the exercise. All the subjects were anesthetized by intraperitoneal injection of chloral hydrate of 10%. The blood was taken through the abdominal aorta and the cryogenic refrigerator was preserved after the centrifugation. In addition, the rectus femoris muscle was separated quickly, The muscle specimens were frozen with liquid nitrogen cooled isopentane and then placed on dry ice to be preserved in ultra low temperature refrigerator after volatilization of isopentane. The samples taken as follows: 1) histomorphology analysis was carried out by freezing section of the rectus femoris HE staining (crosscutting); 2) the colorimetric method and the ELISA Kit were used respectively. Serum CK and CK-MM levels were detected; 3) RT-PCR test was used to detect Myo D, Myogenin m RNA level; 4) Myo D, and the expression of Myogenin protein was detected by Western-Blot experiment. The cell nucleus can be seen at the edge of the musculi membrane. The tissue section results from different time groups after exercise show that the muscle fibers are not arranged closely, the gap becomes larger, the shape is irregular and swollen. The swelling of the muscle fiber is found in the transverse section of the rectus femoris in group 6h after exercise, and the muscle fiber swelling in group 12h after exercise to 48h group after exercise. After exercise, the 48 groups and 72h groups after exercise showed that the muscle fibers were most scattered, the space was the most scattered, the gap was the largest, and the nuclear spillover was the most. The slices of 1W and 2W groups after exercise showed that the distribution of muscle fibers tended to be normal, and the muscle fibers were closely arranged in.2. serum CK and CK-MM: serum CK results showed that the index was in transport. After exercise, group 0h, group 6h, 72h group after exercise and 1W group after exercise were significantly different from those in control group, and the expression level of 48h group was increasing in 0h group after exercise after exercise. The result of serum CK-MM showed that the index was in 24h group after exercise, and there was a statistically significant difference between the 48h group and the control group after exercise. After exercise, the expression level of the 24h group was increasing in group 0h and after exercise, then the decline trend of.3.Myo D, Myogenin m RNA expression changes: the RT-PCR experimental results showed that Myo D m RNA expression level after exercise, after exercise group, after exercise and after exercise, there were statistical significance compared with the control group. Difference (P0.05); Myogenin m RNA expression level in group 12h after exercise, 24h group after exercise and after exercise 48h group compared with control group, there was significant difference (P0.05), Id1 m RNA expression level in 6h group and after exercise after exercise compared with the control group compared with the control group has statistical significant difference. After the downhill running, the Western blot results of Myogenin protein showed that there was a significant difference between the 6h group after exercise and the 12h group after exercise (P0.05), and the same was immediately after the exercise. After the exercise, the 24h group reached the maximum value, then continued down to the pre exercise level. Myo D eggs after the one-off downhill running. The results of white Western blot showed that after exercise, group 0h, group 6h after exercise, 12h group after exercise and 1W group after exercise were significantly different from those in control group (P0.05), and the trend was declining immediately after exercise. After the peak of exercise, the group reached the peak value and continued to decline to the control group. Conclusion 1. a long time downhill running movement was carried out. The experimental model of motor skeletal muscle injury can be successfully induced. 2. in combination with the changes of serum CK and CK-MM, serum CK can be used as a sensitive index for evaluating the damage of athletic skeletal muscle. Serum CK-MM is more accurate as a auxiliary evaluation of motor skeletal muscle injury; 3. Myo D and Myogenin in the injury of skeletal muscle induced by one-off downhill running. The phase difference exists at the transcriptional level.
【学位授予单位】：上海体育学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R87

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