大鼠骨骼肌钝挫伤恢复过程中线粒体融合—分裂基因表达情况的研究

发布时间：2018-04-30 09:35

  本文选题：腓肠肌 + 钝挫伤　； 参考：《山东体育学院》2015年硕士论文

【摘要】：研究目的:创建大鼠腓肠肌钝挫伤实验模型,观测大鼠钝挫伤后线粒体融合-分裂基因的表达情况,探讨大鼠腓肠肌钝挫伤修复过程中线粒体分裂与融合基因表达,阐明腓肠肌损伤后自体修复中线粒体质量控制机制。研究意义:为下一步从能量代谢角度探讨腓肠肌损伤修复限制因素提供理论基础,为之后研究外源性调控损伤修复速度具有重要意义。研究方法:把60只雄性的健康Wistar大鼠随机的分为8个组别,分别是12h组、2d组、5d组、7d组、10d组、15d组、30d组、对照组,用自制钝挫伤打击器对前七组Wistar大鼠的两条后腿的腓肠肌进行打击。前七组依次在大鼠经过钝挫伤打击后的12小时、2天、5天、7天、10天、15天、30天时即刻取材,对照组与12小时组同一天取材,取大鼠两后腿的腓肠肌,提取总RNA。应用RT-PCR技术,以总RNA作为模板,先进行反转录通过使用逆转录酶把RNA反转录成为c DNA,再以c DNA作为模板进行PCR的扩增反应,从而得到大鼠线粒体融合-分裂基因的表达情况。研究结果:1、12h、2d、5d、7d、10d、15d、30d各组的线粒体融合-分裂基因的表达情况的总体变化趋势表现为:各组与正常对照组相比显著性呈现出逐渐降低的趋势,单看各组间的总体表达量情况则呈现上升的趋势。总趋势中2d组、10d组两个阶段各基因的表达量在总体上升的趋势中均出现了突然下降的情况,2d的基因表达量达到最低值,15d、30d的基因表达量处于最高值。2、在30d时,线粒体融合基因的表达量均低于正常对照组,线粒体分裂基因DPR1高于正常对照组,Fist1低于正常对照组。3、线粒体融合基因在12h、2d、5d、10d与对照组相比变化都十分显著,均显著下降;线粒体分裂基因仅在2d变化十分显著。研究结论:1、线粒体融合-分裂基因的表达量在2d达到最低值,处于炎症反应阶段,线粒体呼吸受抑制,融合-分裂功能下降,融合基因的表达量减少导致供能不足,恢复速率缓慢。线粒体融合分裂基因的表达量均在10d下降的机制尚不明确。2、线粒体融合分裂基因的表达情况总体趋势上一致,但线粒体分裂基因的表达量稍高于融合基因的表达情况,主要由线粒体分裂基因负责调控细胞凋亡和能量的重新分配。线粒体的形态趋于分裂可能是线粒体呼吸功能的下降造成的,同时线粒体融合基因减少,线粒体分裂基因增多有利于提高代谢效率,为新生细胞提供足够的能量。3、在30天时线粒体融合分裂基因的表达量趋向于正常水平但总体仍低于正常水平,在修复期损伤部位的纤维组织和生肌组织同一时间进行增生,此时线粒体融合水平差能量供应不足,使得损伤部位没办法恢复正常水平状态,线粒体融合与分裂的动态平衡没有达到最佳水平。线粒体融合-分裂的高度动态平衡对于细胞积极有效地适应和应对新环境的变化情况具有重要作用,有利于损伤后的恢复。
[Abstract]:Objective: to establish an experimental model of blunt gastrocnemius contusion in rats, observe the expression of mitochondrial fusion and mitotic gene after blunt injury of rat gastrocnemius, and investigate the expression of mitochondrial division and fusion gene during the repair of blunt injury of gastrocnemius muscle in rats. To elucidate the mechanism of mitochondrial quality control in autorepair of gastrocnemius after injury. Research significance: to provide a theoretical basis for the further study of the limiting factors of gastrocnemius injury repair from the point of view of energy metabolism, it is of great significance to study the exogenous regulation of injury repair speed. Methods: sixty healthy male Wistar rats were randomly divided into 8 groups: 12 h group, 2 d group, 5 d group, 7 d group, 10 d group, 15 d group, 30 d group, control group. The gastrocnemius muscles of the first seven groups of Wistar rats were attacked with a blunt contusion instrument. The first seven groups were collected immediately after blunt contusion for 12 hours, 7 days, 10 days, 15 days and 30 days. The control group and the 12 hour group were taken from gastrocnemius muscle on the same day, and the total RNA was extracted from the gastrocnemius muscle of both hind legs of the rats. Using RT-PCR technique and total RNA as template, reverse transcription of RNA into c DNA was carried out by reverse transcriptase, then PCR amplification reaction was performed with c DNA as template, and the expression of rat mitochondrial fusion and mitotic gene was obtained. The results showed that the overall trend of mitochondrial fusion and mitotic gene expression in each group was as follows: compared with the control group, the expression of mitochondrial fusion and mitotic gene in each group showed a decreasing trend. The total expression of each group showed an upward trend. In the general trend, the expression of genes in the two stages of 2d group / 10d group showed a sudden decrease in the overall upward trend. The gene expression level of 2d group reached the lowest value at 15d / 30d, and the gene expression level was at the highest value 路2d, and at 30d, the gene expression level of 2d group was at the highest value, and at 30d, the gene expression level of 2d group was at the highest level. The expression of mitochondrial fusion gene was lower than that of normal control group, and the DPR1 of mitotic gene was higher than that of normal control group. The expression of mitochondrial fusion gene was significantly lower than that of control group. Mitochondrial mitotic genes changed significantly only at 2 days. Conclusion: the expression of mitochondrial fusion-mitotic gene reached the lowest level in 2 days. In the stage of inflammation, mitochondrial respiration was inhibited, fusion-division function was decreased, and the expression of fusion gene was decreased, which resulted in insufficient energy supply. The recovery rate is slow. The mechanism of the decrease of mitochondrial fission gene expression at 10 days is unclear. The general trend of mitochondrial fusion cleavage gene expression is consistent, but the expression level of mitochondrial fission gene is slightly higher than that of fusion gene expression. Mitochondrial mitotic genes are responsible for regulating apoptosis and energy redistribution. The decline of mitochondrial respiratory function may be the cause of mitochondrial morphology tending to divide, and the decrease of mitochondrial fusion gene and the increase of mitochondrial fission gene are beneficial to the increase of metabolic efficiency. After 30 days, the expression of Mitochondrial fusion mitotic gene tended to be normal but still below normal level, and proliferated at the same time in the fibrous tissue and muscle tissue of the damaged site during the repair period. At this time, the energy supply of mitochondrial fusion level difference is insufficient, which makes the damaged site unable to return to the normal level, and the dynamic balance between mitochondrial fusion and division does not reach the optimal level. The high dynamic balance of mitochondrial fusion and division plays an important role in adapting to and coping with the changes of the new environment actively and effectively, and is beneficial to the recovery after injury.
【学位授予单位】：山东体育学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：G804.7
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本文编号：1824026