本文選題：創(chuàng)傷 + 心肌損傷　； 參考：《中國人民解放軍軍醫(yī)進(jìn)修學(xué)院》2008年碩士論文

【摘要】： 背景與目的:應(yīng)激是機(jī)體對(duì)有害刺激(應(yīng)激原)所做出的適應(yīng)性綜合反應(yīng)。機(jī)體在受到外界刺激如燒傷、缺血再灌注以及手術(shù)等創(chuàng)傷應(yīng)激后,可以引起一系列神經(jīng)內(nèi)分泌的激活、氧化應(yīng)激以及細(xì)胞內(nèi)的Ca~(2+)超載,誘導(dǎo)體內(nèi)產(chǎn)生大量的血管緊張素Ⅱ(AngⅡ)、醛固酮(Ald)、內(nèi)皮素(ET-1)以及各種細(xì)胞因子和活性氧自由基,從而引起應(yīng)激性高血壓、應(yīng)激性心律失常、應(yīng)激性心肌缺血、心功能障礙等。肢體創(chuàng)傷也是一種應(yīng)激狀態(tài),心血管系統(tǒng)是多種應(yīng)激因素作用的重要靶器官。 本實(shí)驗(yàn)用手術(shù)的方法建立大鼠的左后肢截肢創(chuàng)傷應(yīng)激模型,動(dòng)態(tài)檢測(cè)血漿H_2S、NO、髓過氧化物酶(myeloperoxidase,MPO)、丙二醛(MDA)、血管緊張素Ⅱ(angiotensinⅡ,AngⅡ)、醛固酮(aldosterone,ALD)的變化,檢測(cè)心肌CSE、MPO活性及MDA、ALD含量,并用RT-PCR的方法檢測(cè)心肌CaN mRNA的表達(dá),觀察心肌組織CaN Aβ基因表達(dá)的變化,同時(shí)光鏡下觀察心肌的病理損傷,電鏡觀察心肌線粒體形態(tài)和結(jié)構(gòu)的變化。并應(yīng)用H_2S供體NaHS、CSE抑制劑PPG、CaN抑制劑FK506及醛固酮拮抗劑螺內(nèi)酯進(jìn)行干預(yù),觀察上述指標(biāo)及線粒體呼吸功能、膜電位及線粒體總ATP酶的變化,進(jìn)一步研究H_2S、CaN及醛固酮在肢體創(chuàng)傷應(yīng)激后心肌損傷中的信號(hào)傳導(dǎo)機(jī)制及各信號(hào)通路之間的相互作用,同時(shí)研究H_2S、CaN抑制劑FK506及醛固酮受體拮抗劑螺內(nèi)酯對(duì)心血管系統(tǒng)的保護(hù)作用。 本研究分為三部分:一、截肢創(chuàng)傷應(yīng)激后心肌H_2S/CSE體系和CaN的動(dòng)態(tài)變化及干預(yù)措施:二、截肢創(chuàng)傷應(yīng)激后心肌損傷相關(guān)信號(hào)轉(zhuǎn)導(dǎo)機(jī)制的研究;三、截肢創(chuàng)傷應(yīng)激后心肌損傷的線粒體機(jī)制。 方法:本實(shí)驗(yàn)用手術(shù)的方法建立大鼠的左后肢截肢創(chuàng)傷應(yīng)激模型。第一部分實(shí)驗(yàn)分組:雄性Wistar大鼠63只,隨機(jī)分為9組,每組7只:正常對(duì)照組;截肢后1h組;2h組;4h組;6h組;12h組;24h組;48h組;72h組。第二部分實(shí)驗(yàn)分組:雄性Wistar大鼠42只,分為6組,每組7只:正常對(duì)照組;創(chuàng)傷對(duì)照組;NaHS組:截肢后立即給予腹腔注射NaHS(28μmol/kg);PPG組:截肢后立即給予腹腔注射PPG(50mg/kg);FK506組:截肢后立即給予腹腔注射FK506(0.1mg/kg);螺內(nèi)酯組:正常大鼠螺內(nèi)酯每天灌胃(20mg/kg),6天后截肢。觀察各組大鼠心肌病理、血漿H_2S、NO、MPO、MDA、AngⅡ、ALD的變化,同時(shí)檢測(cè)各組大鼠心肌CSE活性、MPO、MDA、ALD的含量;并用RT-PCR的方法檢測(cè)心肌CaN mRNA的表達(dá),電鏡觀察心肌線粒體形態(tài)結(jié)構(gòu)的變化。第三部分實(shí)驗(yàn)動(dòng)物為48只雄性SD大鼠,隨機(jī)分為6組,每組8只:分組及處理同第二部分。觀察心肌線粒體呼吸功能和膜電位的變化,測(cè)定線粒體總ATP酶。 結(jié)果:第一部分結(jié)果:與正常對(duì)照組比較,血漿MPO、MDA在截肢后1h即有明顯升高,至截肢后6h達(dá)高峰,其后雖有所下降,但仍維持在較高水平。心肌MPO及MDA較血漿變化稍晚,于截肢創(chuàng)傷應(yīng)激后2h開始升高,心肌MPO于12h最高,MDA于截肢后6h最高。血漿H_2S及NO均于創(chuàng)傷應(yīng)激后4h開始明顯下降,6h最低,至創(chuàng)傷后24h逐漸恢復(fù)。而心肌CSE酶活性的變化稍晚于血漿H_2S,于創(chuàng)傷后12h最低,72h后才逐漸恢復(fù)。第二部分結(jié)果:與創(chuàng)傷對(duì)照組比,NaHS組及FK506組血漿及心肌MDA、ALD、H_2S、NO含量增加,PPG組血漿MDA、ALD、H_2S、NO及心肌CSE酶活性均下降,螺內(nèi)酯組血漿H_2S、NO含量增加而心肌CSE酶活性變化不明顯。第三部分結(jié)果:與正常對(duì)照組比,創(chuàng)傷組線粒體呼吸功能下降,RCR及P/O明顯降低,ATP酶含量及膜電位下降。與創(chuàng)傷組比較,NaHS組及FK506組心肌線粒體呼吸功能明顯改善,RCR、P/O及ATP酶含量增加,膜電位升高,而PPG組RCR、P/O及ATP酶含量減少。與創(chuàng)傷對(duì)照組比,螺內(nèi)酯組線粒體RCR、ATP及膜電位增加。 結(jié)論:截肢創(chuàng)傷應(yīng)激后可以造成遠(yuǎn)隔心肌及線粒體損傷,截肢創(chuàng)傷應(yīng)激后4～6小時(shí),心肌損傷最重。炎細(xì)胞及炎性細(xì)胞因子的活化、過度的氧化應(yīng)激、CaN信號(hào)通路及內(nèi)源性血漿H_2S/心肌CSE體系均參與了截肢創(chuàng)傷應(yīng)激后心肌損傷。外源性H_2S補(bǔ)充及CaN抑制劑FK506可以通過降低炎細(xì)胞浸潤及炎性細(xì)胞因子激活、直接清除氧自由基、增加心肌線粒體呼吸功能、提高膜電位及線粒體ATP酶產(chǎn)量,對(duì)心肌損傷發(fā)揮保護(hù)作用。CaN信號(hào)通路及氣體信號(hào)通路相互作用共同調(diào)節(jié)截肢創(chuàng)傷應(yīng)激后的心肌及線粒體損傷。
[Abstract]:Background and purpose: stress is an adaptive comprehensive response to harmful stimuli (Ying Jiyuan). The body can induce a series of neuroendocrine activation, oxidative stress, and intracellular Ca~ (2+) overload after external stimuli such as burns, ischemia reperfusion and surgery, and induce a large number of blood vessels in the body. Tension II (Ang II), aldosterone (Ald), endothelin (ET-1) as well as various cytokines and reactive oxygen radicals can cause stress hypertension, stress arrhythmia, stress myocardial ischemia, cardiac dysfunction, and so on. Limb trauma is also a stress state, and cardiovascular system is an important target organ for various stress factors.
In this experiment, the rat's left hind limb amputation stress model was established by operation, and the changes of plasma H_2S, NO, myeloperoxidase (MPO), malondialdehyde (MDA), angiotensin II (angiotensin II, Ang II), aldosterone (aldosterone, ALD), and CSE, MPO activity and MDA, content of the myocardium were detected. The expression of CaN mRNA in myocardium was detected and the changes of CaN A beta gene expression in myocardial tissue were observed. At the same time, the pathological damage of myocardium was observed under light microscope. The changes of the morphology and structure of myocardial mitochondria were observed by electron microscope. The intervention of H_2S donor NaHS, CSE inhibitor PPG, CaN inhibitor FK506 and aldosterone antagonist spironolactone were used to observe the above indexes and grain lines. The changes of body respiration, membrane potential and mitochondrial ATP enzyme are further studied. The signal transduction mechanism of H_2S, CaN and aldosterone in the myocardial injury after trauma stress and the interaction of various signal pathways are further studied, and the protective effects of H_2S, CaN inhibitor FK506 and aldosterone receptor antagonist spironolactone on cardiovascular system are also studied.
This study is divided into three parts: first, the dynamic changes of H_2S/CSE system and CaN after the amputation of the amputation stress and the intervention measures: two, the study of the signal transduction mechanism of myocardial injury after the amputation stress; three, the mitochondrial mechanism of myocardial injury after the amputation stress.
Methods: the first part of the experiment was to establish the left hind limb amputation trauma stress model in rats. 63 male Wistar rats were divided into 9 groups randomly, 7 rats in each group: normal control group, group 1h after amputation; group 2H; group 4H; 6h group; 12h group; 24h group; 48H group; 72h group. 42 male Wistar rats were divided into 6 groups. 7 rats in each group: normal control group, trauma control group and group NaHS: immediately after amputation, NaHS (28 mol/kg) was given by intraperitoneal injection; group PPG: PPG (50mg/kg) was given immediately after amputation; group FK506: FK506 (0.1mg/kg) immediately after amputation; spironolactone group: normal rat spironolactone was intragastric daily (20mg/kg), and 6 days after amputation. Observe groups large The changes in plasma H_2S, NO, MPO, MDA, Ang II, ALD were observed in rat myocardium, and the content of CSE activity, MPO, MDA and ALD were detected in each group, and the expression of myocardial CaN was detected by RT-PCR method and changes in the morphological structure of myocardial mitochondria were observed by electron microscope. The third experimental animals were randomly divided into 6 groups, 8 rats in each group. Group and treatment of the same second parts. The changes of respiratory function and membrane potential of mitochondria were observed, and the total ATP enzyme of mitochondria was determined.
Results: the first part results: compared with the normal control group, the plasma MPO and MDA increased significantly after the amputation, and the 6h reached the peak after amputation. After the amputation, the 6h reached a high level, but still maintained at a higher level. The myocardial MPO and MDA were slightly later than the plasma changes, and the 2H began to rise after the amputation stress, and the heart MPO was the highest in 12h, and MDA was the highest after the amputation. Plasma 6h was the highest after amputation. Both H_2S and NO began to decrease obviously after the trauma stress, and the lowest 6h, and the 24h gradually recovered after the trauma. The change of myocardial CSE activity was slightly later than that of plasma H_2S, and 12h was lowest after trauma, and gradually recovered after 72h. The second part: compared with the trauma control group, the plasma and FK506 group of NaHS and FK506 groups were increased. The activity of ALD, H_2S, NO and myocardial CSE decreased, the plasma H_2S, NO content of spironolactone group increased and the myocardial CSE enzyme activity was not significantly changed. Third results: compared with the normal control group, the mitochondrial respiratory function decreased, RCR and P/O decreased significantly, the ATP enzyme content and membrane potential decreased. Compared with the trauma group, NaHS group and FK506 group myocardial mitochondria granules were compared. The volume of body respiration was obviously improved, the content of RCR, P/O and ATP increased, the membrane potential was increased, while the content of RCR, P/O and ATP in group PPG decreased. Compared with the control group, the mitochondrial RCR, ATP and membrane potential of the spironolactone group were increased.
Conclusion: the injury of the amputation injury can cause the injury of the distant myocardium and mitochondria, the most serious injury of the myocardium is 4~6 hours after the amputation stress. The activation of the inflammatory cells and inflammatory cytokines, the excessive oxidative stress, the CaN signaling pathway and the endogenous plasma H_2S/ myocardial CSE system all participate in the myocardial injury after the amputation stress. Exogenous H_2S Supplementation and CaN inhibitor FK506 can directly remove oxygen free radicals, increase myocardial mitochondrial respiratory function, increase membrane potential and mitochondrial ATP enzyme production by reducing infiltration of inflammatory cells and inflammatory cytokines activation, and improve the membrane potential and mitochondrial ATP enzyme production. The protective effect of.CaN signaling pathway and gas signal pathway interaction to regulate amputation stress stress on myocardial injury Injury of the posterior myocardium and mitochondria.
【學(xué)位授予單位】：中國人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R363

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