大鼠骨骼肌损伤后Nrf-2、HO-1蛋白的表达及作用机制研究

发布时间：2018-11-19 12:44

【摘要】：目的:通过研究Nrf-2、HO-1蛋白在大鼠骨骼肌损伤修复过程中的分布及表达变化规律,探索其在骨骼肌损伤修复过程中的作用机制及其表达变化与损伤时间的关系。方法:本研究使用10~12周龄的Sprague Dawley雄性大鼠腓肠肌内注射心脏毒素(Cardiotoxin,CTX)制作骨骼肌损伤模型,分别取损伤后多个时间点(0h、1h、4h、8h、12h、16h、1d、3d、5d、7d、9d、13d、17d、21d)的损伤处组织,采用HE染色法观察骨骼肌损伤修复过程中的形态学变化,用DCFH-DA探针法检测活性氧(Reactive oxygen species,ROS)含量,用Western blotting及免疫荧光染色检测Nrf-2、HO-1蛋白表达水平,用实时荧光定量PCR(Quantitative Real-time PCR)法检测Nrf-2m RNA表达水平。结果:注射CTX损伤大鼠骨骼肌后,损伤区肌细胞迅速水肿、坏死,随后相继出现中性粒细胞、单核细胞为主的炎细胞浸润,第5天大量新生肌细胞生成,至第21天基本完成肌细胞修复;与对照组比较,骨骼肌损伤早期(1小时内),ROS出现先升后降的迅速变化;Nrf-2 m RNA及蛋白仅少数几组表达增多,而Nrf-2蛋白核转位在损伤后第1小时即显著升高,在损伤后第1天核内表达最高,随后逐渐恢复到对照组水平;HO-1蛋白表达量在骨骼肌CTX损伤后也迅速增高,在第1到3天达高峰,随后逐渐减少,最后恢复到对照水平。实验观察到骨骼肌损伤后Nrf-2蛋白阳性细胞核率及HO-1表达量具有正线性相关,二者均随损伤时间呈时序性变化。结论:1.氧化应激信号通路参与调控大鼠骨骼肌损伤后的修复过程,Nrf-2及HO-1蛋白通过抑制过度的炎症反应、激活肌卫星细胞,诱导其增殖及分化以及新生肌管的融合,促进损伤骨骼肌结构及功能的恢复。2.大鼠骨骼肌在注射CTX损伤后的修复过程中,Nrf-2蛋白主要通过核转位,增加核内Nrf-2蛋白含量实现其调控作用,并非通过大量合成新Nrf-2蛋白。3.在大鼠骨骼肌损伤后的修复过程中,Nrf-2蛋白阳性细胞核率及胞浆内HO-1蛋白含量随损伤时间呈时序性变化,可以作为损伤时间推断的新指标。
[Abstract]:Aim: to study the distribution and expression of Nrf-2,HO-1 protein in the course of skeletal muscle injury and repair in rats, and to explore the mechanism of Nrf-2,HO-1 protein expression in the process of skeletal muscle injury repair and the relationship between the expression change and injury time. Methods: the model of skeletal muscle injury was established by intramuscular injection of cardiac toxin (Cardiotoxin,CTX) in 10 ~ 12 weeks old Sprague Dawley male rats. HE staining method was used to observe the morphological changes during skeletal muscle injury repair, DCFH-DA probe method was used to detect the content of reactive oxygen (Reactive oxygen species,ROS (Ros), and Western blotting and immunofluorescence staining were used to detect Nrf-2,. The expression level of HO-1 protein was detected by real-time fluorescence quantitative PCR (Quantitative Real-time PCR. Results: after CTX was injected into the skeletal muscle of rats, the muscle cells in the injured area were rapidly edema and necrosis, and then neutrophil, monocyte mainly inflammatory cells infiltrated, and on the 5th day, a large number of neomyocytes were formed. On the 21st day, the repair of myocytes was basically completed. Compared with the control group, there was a rapid change of), ROS in the early stage of skeletal muscle injury (), ROS increased first and then decreased within 1 hour). The expression of Nrf-2 m RNA and protein increased in only a few groups, but the Nrf-2 protein nuclear translocation increased significantly at 1 hour after injury, and reached the highest level on the first day after injury, and then gradually returned to the level of control group. The expression of HO-1 protein increased rapidly after CTX injury of skeletal muscle, peaked at the 1st to 3rd day, then decreased gradually, and finally returned to the control level. It was observed that there was a positive linear correlation between the positive nuclear rate of Nrf-2 protein and the expression of HO-1 after skeletal muscle injury. Conclusion: 1. Oxidative stress signaling pathway plays an important role in the repair of skeletal muscle injury in rats. Nrf-2 and HO-1 proteins stimulate the proliferation and differentiation of muscle satellite cells by inhibiting excessive inflammatory response, and induce the fusion of newborn muscle tubes. Promote the recovery of skeletal muscle structure and function. 2. During the repair of rat skeletal muscle after injection of CTX, Nrf-2 protein was regulated mainly by nuclear translocation and increased the content of Nrf-2 protein in the nucleus, but not by the synthesis of a large number of new Nrf-2 proteins. During the repair of rat skeletal muscle after injury, the positive nuclear rate of Nrf-2 protein and the content of HO-1 protein in the cytoplasm showed a temporal change with the injury time, which could be used as a new index for estimating the injury time.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：D919

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