【摘要】：目的:通過研究Nrf-2、HO-1蛋白在大鼠骨骼肌損傷修復過程中的分布及表達變化規(guī)律,探索其在骨骼肌損傷修復過程中的作用機制及其表達變化與損傷時間的關系。方法:本研究使用10~12周齡的Sprague Dawley雄性大鼠腓腸肌內(nèi)注射心臟毒素(Cardiotoxin,CTX)制作骨骼肌損傷模型,分別取損傷后多個時間點(0h、1h、4h、8h、12h、16h、1d、3d、5d、7d、9d、13d、17d、21d)的損傷處組織,采用HE染色法觀察骨骼肌損傷修復過程中的形態(tài)學變化,用DCFH-DA探針法檢測活性氧(Reactive oxygen species,ROS)含量,用Western blotting及免疫熒光染色檢測Nrf-2、HO-1蛋白表達水平,用實時熒光定量PCR(Quantitative Real-time PCR)法檢測Nrf-2m RNA表達水平。結(jié)果:注射CTX損傷大鼠骨骼肌后,損傷區(qū)肌細胞迅速水腫、壞死,隨后相繼出現(xiàn)中性粒細胞、單核細胞為主的炎細胞浸潤,第5天大量新生肌細胞生成,至第21天基本完成肌細胞修復;與對照組比較,骨骼肌損傷早期(1小時內(nèi)),ROS出現(xiàn)先升后降的迅速變化;Nrf-2 m RNA及蛋白僅少數(shù)幾組表達增多,而Nrf-2蛋白核轉(zhuǎn)位在損傷后第1小時即顯著升高,在損傷后第1天核內(nèi)表達最高,隨后逐漸恢復到對照組水平;HO-1蛋白表達量在骨骼肌CTX損傷后也迅速增高,在第1到3天達高峰,隨后逐漸減少,最后恢復到對照水平。實驗觀察到骨骼肌損傷后Nrf-2蛋白陽性細胞核率及HO-1表達量具有正線性相關,二者均隨損傷時間呈時序性變化。結(jié)論:1.氧化應激信號通路參與調(diào)控大鼠骨骼肌損傷后的修復過程,Nrf-2及HO-1蛋白通過抑制過度的炎癥反應、激活肌衛(wèi)星細胞,誘導其增殖及分化以及新生肌管的融合,促進損傷骨骼肌結(jié)構(gòu)及功能的恢復。2.大鼠骨骼肌在注射CTX損傷后的修復過程中,Nrf-2蛋白主要通過核轉(zhuǎn)位,增加核內(nèi)Nrf-2蛋白含量實現(xiàn)其調(diào)控作用,并非通過大量合成新Nrf-2蛋白。3.在大鼠骨骼肌損傷后的修復過程中,Nrf-2蛋白陽性細胞核率及胞漿內(nèi)HO-1蛋白含量隨損傷時間呈時序性變化,可以作為損傷時間推斷的新指標。
[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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：D919

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