本文關鍵詞： 骨骼肌鈍挫傷 線粒體自噬 骨骼肌超微結構 缺氧 AMPKα2 HIF-1α BNIP3 NIX 活性氧 線粒體電子傳遞鏈酶復合體 ATP合成酶　出處：《上海體育學院》2016年博士論文　論文類型：學位論文

【摘要】：研究目的:骨骼肌鈍挫傷發(fā)生后,受損骨骼肌除了發(fā)生經典的血管反應、炎癥反應外,其內部還會形成缺氧環(huán)境,這種環(huán)境形成后可能引起線粒體功能降低,導致ATP產生不足,最終勢必會使損傷的恢復時程延長,功能恢復不完全,甚至喪失正常解剖結構,預后較差�，F有研究已證實,線粒體自噬是維持線粒體質量的重要力量,也是機體對代謝改變做出的一種重要的代償性反應,該過程可及時清除損傷、功能障礙的線粒體,從而減少ROS的生成,而過多的ROS會直接損害健康線粒體,那么及時清除破損線粒體就可以防止損傷范圍擴大。根據上述依據提出假設:1.損傷后骨骼肌超微結構發(fā)生改變:線粒體形態(tài)改變、完整性缺失,可見到線粒體自噬體的存在;2.鈍挫傷發(fā)生后在骨骼肌恢復過程中ROS水平增高,線粒體功能下降;3.在觀察骨骼肌鈍挫傷恢復時程內,線粒體自噬被誘導且在損傷早期最為顯著,而線粒體功能下降也發(fā)生在這一時期。本研究用重物砸傷動物骨骼肌制作動物模型,模擬運動中的骨骼肌鈍挫傷,使用透射電鏡觀察線粒體自噬體及數量、檢測受損組織中ROS生成量、對比線粒體呼吸鏈酶復合體活性及線粒體自噬相關指標的時程性變化等,通過這些指標的變化來觀測和評價鈍挫傷發(fā)生后線粒體自噬、線粒體功能的改變,從一個嶄新視角來觀察、解釋骨骼肌鈍挫傷發(fā)生、恢復的可能機制,并推測該過程中誘導線粒體自噬發(fā)生可能因素,希望可以為今后骨骼肌鈍挫傷的臨床治療、康復、預防提供更多的理論支持。研究方法:隨機選取64只雄性Wistar大鼠,分為8組:正常對照組以及鈍挫傷后12h、2d、5d、7d、10d、15d、30d取材組(h:小時;d:天),每組8只。除對照組外其他各組用重物砸傷造模后按各組所示時間點取材,檢測腓腸肌AMPKα2、HIF-1α、NIX、BNIP3蛋白、m RNA表達的變化;用透射電鏡觀察鈍挫傷后骨骼肌超微結構的變化,觀察是否存在自噬體及其自噬體數量的時程性改變;檢測ROS、線粒體跨膜電位(ΔΨm)、電子傳遞鏈酶復合體CⅠ-Ⅳ、ATP合成酶等體現線粒體功能指標的時程性改變。通過檢測損傷發(fā)生后上述各指標的變化情況,分析該過程中線粒體功能的改變,以及是否在該過程中發(fā)生了線粒體自噬,并推斷線粒體自噬發(fā)生機制、可能產生的代償后果;線粒體功能是否被損害,推測鈍挫傷損傷線粒體功能降低的可能機制及可能產生的后果。研究結果:1透射電鏡下觀察骨骼肌超微結構在骨骼肌鈍挫傷發(fā)生后時程性改變:從損傷發(fā)生直至傷后30天,均能觀察到被雙層膜包裹、半包裹的線粒體,傷后10天內更易觀察到線粒體自噬體。2代謝相關蛋白AMPKα2、缺氧相關因子HIF-1α在損傷初期都有顯著的升高(p0.05),隨著時間推移,二者表達從傷后5-7d就基本回落到正常水平,直至傷后30d。就二者m RNA表現來看,只有HIF-1α蛋白表達與其m RNA表達不一致。3線粒體自噬相關蛋白BNIP3、NIX表達水平在傷后12h-5d升高明顯(p0.05)HIF-1α表達與二者表現基本接近。此外,在二者水平增高組,電鏡下可觀察到較多數量被溶酶體包裹的線粒體。4鈍挫傷后ΔΨm呈現明顯的時程性改變:在損傷早期(12h、2d、5d)ΔΨm顯著下降(p0.05),該指標改變同BNIP3、NIX表達增高所在時間段基本吻合。5損傷后12h-5d各組ROS與對照組相比均有升高,且差異顯著(p0.05)。6損傷后12h-5d,線粒體電子傳遞鏈酶復合體C1、CⅢ活性升高,與對照組相比差異顯著(p0.05),這也同ROS水平的升高發(fā)生在相同時間段,而另外兩個酶復合體活性并未發(fā)生顯著變化。ATP合成酶的活性在損傷早期下降明顯(p0.05),從傷后7d開始逐漸回升至正常水平并保持到傷后30d。結論:1高濃度的ROS可直接攻擊線粒體呼吸鏈造成線粒體損傷,而產生內源性ROS的部位又恰好是在線粒體電子傳遞鏈上。ROS升高的階段與CⅠ、CⅢ活性升高的時間段基本一致,這說明線粒體電子傳遞鏈通過影響CⅠ、CⅢ活性直接對缺氧產生反應,CⅠ、CⅢ又是ROS產生的主要部位,故該階段受損線粒體產生大量ROS,而體內的ROS清除系統(tǒng)無法及時清除過多的ROS。2代謝調節(jié)關鍵因子AMPKα2、缺氧相關因子HIF-1α均在線粒體功能變化期間出現高表達,該階段更易觀察到線粒體自噬體,這說明二者可能從對合成分解代謝的調整和線粒體質量控制方面做出代償性調節(jié)。3線粒體自噬相關蛋白BNIP3、NIX在線粒體功能下降期間出現明顯的高表達,同時透射電鏡觀察結果顯示該階段受損骨骼肌也存在許多被溶酶體全包裹或半包裹的線粒體。這說明在損傷后線粒體功能下降期間發(fā)生了線粒體自噬,且參與了機體的調整過程,通過對受損線粒體的清除,達到減少ROS的產生的目的,防止損傷范圍擴大。雖然過度的線粒體自噬會引起細胞的凋亡,但就本實驗來看,傷側肢體功能是逐漸恢復的,且并未出現骨骼肌的壞死,說明線粒體自噬程度并未過度,作用是積極的。4正常線粒體跨膜電位(ΔΨm)的維持,是線粒體能否維持功能的關鍵,該指標的變化也是發(fā)生線粒體自噬和細胞凋亡的重要條件。在鈍挫傷骨骼肌恢復過程中,ΔΨm表現出明顯的時程性改變。在損傷早期(12h、2d、5d)ΔΨm顯著下降,該結果結合BNIP3、NIX表達及電鏡觀察結果來推測,損傷后線粒體自噬被誘導,同時對比線粒體功能下降時間段推測線粒體自噬參與到損傷后的代償機制中。
[Abstract]:Objective: To study skeletal muscle contusion, damaged skeletal muscle vascular reaction in addition to the classic, inflammatory reaction, but also the formation of its internal anoxic environment, this environment may cause the formation of reduced mitochondrial function, leading to ATP problems, will eventually be restored to the prolonged injury, incomplete functional recovery, and even loss the normal anatomic structure, poor prognosis. Existing studies have confirmed that mitochondrial autophagy is an important force to maintain mitochondrial quality, an important compensatory response is the body to make metabolic changes, the process can be timely removal of damage, mitochondrial dysfunction, to reduce the production of ROS, and too much ROS will directly harm the health of mitochondria then, the timely removal of damaged mitochondria can prevent the damage scope. According to the above basis for assumptions: 1. after the injury of skeletal muscle ultrastructure change: Mitochondria Body shape, lack of integrity, visible to mitochondrial autophagy; 2. in skeletal muscle contusion after recovery of ROS levels in the process of increased mitochondrial dysfunction; 3. in the recovery of skeletal muscle contusion duration, mitochondrial autophagy was induced and the injury was the most significant, but also a decline in mitochondrial function during this period. The study of animal models with bruise animal skeletal muscle, simulate the movement in skeletal muscle contusion, and the number of autophagosomes were observed using transmission electron microscopy, ROS content detection of damaged tissue, comparing related indicators of mitochondrial respiratory chain enzyme complex activity and mitochondrial autophagy duration changes. The changes in these indicators to monitor and evaluate the contusion after the occurrence of mitochondrial autophagy, mitochondrial function changes, from a new perspective, to explain the occurrence of skeletal muscle contusion, recovery The possible mechanism of the complex, and that induced mitochondrial autophagy in this process may occur, the hope can for clinical treatment in skeletal muscle contusion rehabilitation, prevention to provide more theoretical support. Methods: 64 randomly selected male Wistar rats were divided into 8 groups: normal control group and 12h after contusion 2D, 5D, 7d, 10d, 15d, 30d, material group (h: hours; d: days), 8 rats in each group. Except the control group the other groups with bruise after modeling were shown by time were detected in gastrocnemius AMPK alpha 2, alpha HIF-1, NIX, BNIP3 protein, RNA m the change of expression; transmission electron microscope was used to observe the ultrastructure of skeletal muscle after contusion, observe whether there is any history of the number of autophagosomes and autophagy change detection; ROS, mitochondrial membrane potential (delta m), electron transport chain complexes C I - IV, ATP synthase reflects the history of mitochondrial function index changes. The change of damage detection after the occurrence of the above indicators, analysis of mitochondrial function in the process of change, and whether the occurrence of mitochondrial autophagy in this process, and infer the mechanism of mitochondrial autophagy, the possible consequences of compensation; mitochondrial function has been damaged, speculated that the mechanism of functional damage of mitochondria and may reduce blunt contusion the consequences of the results: 1. Transmission electron microscopy was used to observe the ultrastructure of skeletal muscle in the process of changing the occurrence of skeletal muscle contusion: from the injury until 30 days after injury, were observed by double membrane wrapped mitochondria injury within 10 days after it is easier to study mitochondrial autophagy.2 metabolism related protein AMPK alpha 2, alpha HIF-1 hypoxia related factors are significantly increased in the early stage of injury (P0.05), with the passage of time, the expression of two 5-7d after injury from basic back to normal level, until the 30D. after injury two m RNA performance, only HIF-1 expression and expression of M RNA with.3 mitochondrial autophagy related protein BNIP3, NIX expression level at 12h-5d after injury was significantly increased (P0.05) HIF-1 expression and two basic approaches. In addition, in the two group. The level of observation under electron microscopy the number was wrapped in lysosomal mitochondrial.4 after contusion was only m presents the history of change in the early injury (12h, 2D, 5d) was significantly decreased while m (P0.05), the index change with BNIP3, the increased expression of NIX where the time is consistent with the.5 after injury in 12h-5d group and control ROS compared group were increased, and the difference was significant (P0.05).6 12h-5d after injury, the mitochondrial electron transport chain complexes C1, increased the activity of C III, compared with the control group had significant difference (P0.05), this also with high levels of ROS occurred at the same time, while the other two enzyme complex activity Did not change significantly.ATP synthetase activity decreased significantly in the early injury (P0.05), from 7d after injury began to rise gradually to the normal level and kept to 30D. after injury. Conclusion: high concentration of 1 ROS can directly attack the mitochondrial respiratory chain caused by the injury of mitochondria, and the endogenous ROS of the site is just in the mitochondrial electron transfer stage and C chain of.ROS increased 1 time, increased the activity of C III is basically the same, indicating that the mitochondrial electron transport chain by C I, C III activity directly respond to hypoxia, C I, C III is the main part of ROS, the stage of damaged mitochondria produce large amounts of ROS. The in vivo ROS scavenging system can not be timely cleared many key factors regulating the metabolism of ROS.2 AMPK alpha 2, high expression of hypoxia related factor HIF-1 alpha were changes in mitochondrial function during this stage, it is easier to study mitochondrial autophagy, This shows that the two may make compensatory adjustments and from the mitochondrial quality control synthesis metabolism regulation of.3 mitochondrial autophagy related protein BNIP3, NIX decreased during high expression was evident in mitochondrial function, and transmission electron microscopy results showed that the stage of injured skeletal muscle also has many lysosomal coated or semi wrapped mitochondria. This shows that in the injury of mitochondrial dysfunction occurred during mitochondrial autophagy is involved in the adjustment process, and through the body, for the removal of damaged mitochondria, to reduce the production of ROS to prevent the damage scope. Although excessive apoptosis mitochondrial autophagy leads to cell, but in this experiment, the limb function injury side is gradually recovered, and did not appear in skeletal muscle necrosis, indicating mitochondrial autophagy degree are not excessive, the positive effects of.4 normal mitochondrial transmembrane Potential (a ~ m) maintenance, is the key to maintain mitochondrial function, an important condition for change in the index is mitochondrial autophagy and apoptosis in skeletal muscle contusion in the recovery process, while m showed a history of significant changes. In the early injury (12h, 2D, 5d). While m decreased significantly, the results combined with BNIP3, the expression of NIX and electron microscope results suggest that mitochondrial autophagy was induced after injury, compared with a decline in mitochondrial function time that mitochondrial autophagy involved in the compensatory mechanism of injury.

【學位授予單位】：上海體育學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R873

【參考文獻】

相關期刊論文 前10條

1 姚令良;肖揚;譚華清;;全身炎癥反應綜合征發(fā)病機制理性辨析[J];醫(yī)學與哲學(B);2016年01期

2 李麗君;唐圣松;;BNIP3調控腫瘤細胞自噬及凋亡的研究進展[J];臨床與病理雜志;2014年06期

3 楊朝;，

本文編號：1549305