發(fā)布時間：2018-03-15 01:23

  本文選題：膿毒癥　切入點：心功能損傷　出處：《浙江大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景膿毒癥(sepsis)是由感染引起的全身炎癥反應(yīng)綜合征,膿毒癥的全球發(fā)病率很高。嚴重膿毒癥患者會出現(xiàn)不同程度的心功能下降,并增加患者死亡風(fēng)險。線粒體功能紊亂是膿毒癥時引起心肌細胞不可逆轉(zhuǎn)的損害的主要原因。近年來研究表明,心臟線粒體分裂融合的動態(tài)平衡狀態(tài)在維持心臟線粒體正常生理狀態(tài)中具有重大的作用。而大量研究表明,在心肌缺血再灌注損傷、心力衰竭、高血壓、糖尿病心肌病、動脈粥樣硬化等多種心血管疾病的發(fā)生發(fā)展中,線粒體分裂融合的動態(tài)平衡紊亂起了至關(guān)重要的作用。動力相關(guān)蛋白1(dynamin-related peptide 1,Drp1)是參與線粒體分裂過程中的一種關(guān)鍵蛋白。Drp1是否參與了膿毒癥心肌損傷作用尚不明了。目的本實驗將從整體和細胞水平入手研究膿毒癥時Drp1在心肌損傷中的作用和作用機制;并探討在膿毒癥時,IL-6和TNF-α哪個是導(dǎo)致Drp1改變的主要炎癥因子。方法1)SD大鼠腹腔注射LPS建立膿毒癥模型,測定大鼠生存率。2)心臟超聲檢查測定大鼠心功能。3)提取心肌組織總蛋白和線粒體蛋白,測定各組分中Drp1、Drp1 S637、Drp1 S616、CaMKⅡ、p-JNK、Rac1/Cdc42、RhoA 蛋白水平。4)ELISA法測定大鼠血清中炎癥因子(LPS、TNF-α、IL-6)濃度。5)透射電鏡觀察大鼠心臟線粒體形態(tài)。6)激光共聚焦顯微技術(shù)觀察心肌細胞線粒體形態(tài)。結(jié)果1)SD大鼠腹腔注射不同濃度LPS(5、10、20mg/kg)后,每搏輸出量和心輸出量開始下降;線粒體片段化程度增加。2)SD大鼠給予不同濃度LPS(5、10、20mg/kg)腹腔注射后6h,心肌組織總Drp1水平無明顯變化,但線粒體Drp1量隨著LPS濃度的增高明顯增加。提示,膿毒癥心肌組織中Drp1發(fā)生了線粒體轉(zhuǎn)位。3)SD大鼠腹腔注射LPS(20mg/kg)后,大鼠死亡率達70%;而Drp1的特異性抑制劑Mdivi-1(1 mg/kg,i.p.)可明顯抑制LPS誘導(dǎo)的心肌每搏輸出量和心輸出量的下降,并降低大鼠死亡率。4)不同濃度LPS注射后6h,發(fā)現(xiàn)心肌組織中磷酸化Drp1 S637蛋白水平無明顯變化,但磷酸化Drp1 S616蛋白水平明顯增加。5)SD大鼠腹腔注射LPS(20mg/kg)后,血清中炎癥因子LPS、TNF-α、IL-6濃度明顯增加。LPS(0.01、0.1 或 1 μg/mL)、TNF-α(5、10 或 20ng/mL)、IL-6(5、10或20ng/mL)孵育H9C2細胞48h后,可濃度依賴性降低細胞的生存率。6)LPS(1 μg/mL)、IL-6(20ng/ml)對 H9C2 細胞總 Drp1 和線粒體 drp1 水平無明顯影響。TNF-α(20ng/ml)對H9C2細胞Drp1總量無明顯影響,但TNF-α孵育細胞后,Drp1 Ser616位點的磷酸化和線粒體Drp1水平明顯增加。7)TNF-α(20ng/ml)處理H9C2細胞后,可明顯增加CaMKⅡ的表達。但CaMKⅡ的抑制劑KN-93不能明顯抑制TNF-α誘導(dǎo)的Drp1的磷酸化和線粒體轉(zhuǎn)位,同時對TNF-α誘導(dǎo)的心肌細胞死亡也沒有明顯影響。8)TNF-α(20 ng/ml)處理 H9C2 細胞后,對細胞 p-JNK、Rac1/Cdc42 無明顯影響,但RhoA蛋白表達明顯增加。ROCK1和ROCK2的特異性抑制劑Y-27632和Fasudil可明顯抑制TNF-α的Drp1的磷酸化和線粒體轉(zhuǎn)位,同時有對抗TNF-α降低心肌細胞生存率的作用。激光共聚焦結(jié)果顯示,Y-27632和Fasudil同時可抑制TNF-α誘導(dǎo)的線粒體片段化增多。結(jié)論本實驗結(jié)果表明膿毒癥心肌損傷與Drp1線粒體轉(zhuǎn)位有關(guān),其轉(zhuǎn)位機制可能是由于Drp1 S616發(fā)生了磷酸化。TNF-α可能是膿毒癥心肌損傷時引起Drp1 S616磷酸化的主要炎癥因子,而RohA/ROCK通路可能參與了 TNF-α誘導(dǎo)的Drp1 S616磷酸化和線粒體轉(zhuǎn)位。
[Abstract]:Background sepsis (sepsis) is a systemic inflammatory response syndrome caused by infection, sepsis, the global incidence rate is very high. In patients with severe sepsis can appear heart function decreased in different degrees, and increase the risk of death in patients. Mitochondrial dysfunction is a major cause of myocardial cell irreversible damage in sepsis the study shows that in recent years, the dynamic equilibrium state of heart mitochondrial fission and fusion plays an important role in maintaining the normal physiological state of heart mitochondria. While a large number of studies show that in myocardial ischemia reperfusion injury, heart failure, high blood pressure, diabetic cardiomyopathy, atherosclerosis and other cardiovascular diseases, disorder of dynamic balance of mitochondrial fission fusion has played a crucial role. Dynamin related protein 1 (dynamin-related peptide 1, Drp1) is a key in mitochondrial fission process Protein.Drp1 is involved in myocardial injury in sepsis remains unclear. Objective: this experiment will start on sepsis Drp1 in myocardial injury and the mechanism of the whole and cellular level; and to explore in sepsis, IL-6 and TNF- alpha which is the main inflammatory factors lead to changes in Drp1 method. 1) SD rats were intraperitoneally injected with LPS to establish the model of sepsis rats were survival.2) echocardiography determination of heart function in rats with.3) to extract the total protein and mitochondrial protein in myocardial tissue, determination of Drp1 components in Drp1 S637, Drp1, S616, CaMK II, p-JNK, Rac1/Cdc42, RhoA).4 protein level determination of inflammatory factors in serum of rats by ELISA (LPS, TNF- alpha, IL-6) concentration of.5).6 rat heart mitochondria were observed by transmission electron microscope) observation of mitochondria myocardial cells by confocal laser microscopy. Results 1) intraperitoneal injection of SD in rats with concentration of LPS (5,10,20mg/kg ), stroke volume and cardiac output decreased; mitochondrial fragmentation increased.2) SD rats were treated with different concentrations of LPS (5,10,20mg/kg) after intraperitoneal injection of 6h, no significant changes in total Drp1 level of myocardial tissue, but the amount of mitochondrial Drp1 with increase of the concentration of LPS increased significantly. The results indicated that the occurrence of mitochondrial translocation of.3 Drp1 in myocardial tissue of sepsis) in SD rats by intraperitoneal injection of LPS (20mg/kg), rat mortality rate was 70%; while the Drp1 specific inhibitor Mdivi-1 (1 mg/kg, i.p.) decreased significantly inhibited LPS induced cardiac stroke volume and cardiac output, and reduce the mortality of rats) in different.4 the concentration of LPS after injection of 6h phosphate in myocardial tissue of Drp1 S637 protein level had no obvious change, but the phosphorylation of Drp1 S616 protein level increased.5) SD rats by intraperitoneal injection of LPS (20mg/kg), serum inflammatory cytokines LPS, TNF- alpha, IL-6 concentration significantly increased.LPS (0.01 0.1, or 1 g/mL), alpha TNF- (5,10 or 20ng/mL), IL-6 (5,10 or 20ng/mL) of H9C2 cells incubated with 48h, a concentration dependent decrease in cell survival rate of.6 (LPS) 1 g/mL), IL-6 (20ng/ml) on H9C2 cell total Drp1 and mitochondrial drp1 levels had no significant effect on.TNF- alpha (20ng/ ml) had no obvious effect on the total H9C2 of Drp1 cells, but TNF- alpha after the cells were incubated in Drp1, phosphorylation of Ser616 and mitochondrial Drp1 levels significantly increased.7) TNF- alpha (20ng/ml) in H9C2 cells after treatment, can significantly increase the expression of CaMK II. But the CaMK II inhibitor KN-93 can not inhibit TNF- alpha induced Drp1 phosphorylation and mitochondrial translocation, while TNF- induced myocardial cell death and no obvious effect of.8 TNF- (ng/ml) alpha 20) after treatment of H9C2 cells, the p-JNK cells had no significant effect on Rac1/Cdc42, but significant increase in the expression of RhoA specific inhibitor Y-2763.ROCK1 and ROCK2 2 and Fasudil can significantly inhibit TNF- alpha Drp1 phosphorylation and mitochondrial translocation, and against TNF- alpha decreased myocardial cell survival rate. Confocal laser scanning results showed that mitochondrial Y-27632 fragments and Fasudil also inhibited TNF- induced increased. Conclusion the experimental results show that the myocardial injury in sepsis associated with Drp1 the mitochondrial translocation, translocation may be due to the occurrence of S616 Drp1 phosphorylation of.TNF- alpha may be mainly caused by the inflammatory cytokines Drp1 S616 phosphorylation in septic myocardial injury in sepsis, and RohA/ROCK pathway may be involved in the TNF- induced Drp1 S616 phosphorylation and mitochondrial translocation.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R459.7;R542.2

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