本文關(guān)鍵詞：調(diào)控線粒體形態(tài)與功能恢復(fù)高糖狀態(tài)下七氟醚后處理心肌保護(hù)作用的機(jī)制研究　出處：《新疆醫(yī)科大學(xué)》2017年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 心肌保護(hù) 線粒體動(dòng)力學(xué) 分裂與融合 糖尿病 七氟醚后處理

【摘要】：目的:心血管疾病嚴(yán)重威脅圍術(shù)期麻醉與手術(shù)安全。缺血再灌注損傷(IRI)是圍術(shù)期心臟不良事件發(fā)生的主要原因。通過(guò)在再灌注早期應(yīng)用七氟醚實(shí)現(xiàn)心肌保護(hù)效應(yīng)的方法被稱(chēng)為七氟醚后處理(Sevoflurane Postconditionning,SPostC)。SPostC可產(chǎn)生類(lèi)似缺血/缺氧預(yù)處理的心肌保護(hù)作用,且已經(jīng)證實(shí)對(duì)于年輕健康的心肌所發(fā)生的IRI具有良好的心肌保護(hù)效果,是極具希望的抗心肌損傷的治療手段。然而,糖尿病狀態(tài)下,SPostC心肌保護(hù)效果缺失或弱化,嚴(yán)重制約了SPostC的轉(zhuǎn)化應(yīng)用。并且使用胰島素使糖尿病大鼠血糖水平正常后仍不能恢復(fù)SPostC的心肌保護(hù)作用。如何才能恢復(fù)現(xiàn)有心肌保護(hù)手段對(duì)糖尿病等病理性心肌的保護(hù)作用,是目前圍術(shù)期醫(yī)學(xué)亟待解決的重要臨床問(wèn)題。本研究期望通過(guò)探討七氟醚后處理的線粒體保護(hù)機(jī)制,尋找保護(hù)效應(yīng)弱化的關(guān)鍵,并觀察高糖對(duì)七氟醚后處理調(diào)控線粒體融合分裂作用的干擾與影響;最終通過(guò)調(diào)控線粒體融合分裂平衡恢復(fù)高糖狀態(tài)下SPostC的心肌保護(hù)作用。方法:建立在體大鼠IRI模型與離體Langendorff心臟缺血再灌模型。在體大鼠IRI模型,缺血30min再灌注2h,測(cè)定心肌梗死面積、ATP含量與JAK2、STAT3的磷酸化水平。離體Langendorff心臟缺血再灌模型,復(fù)灌末測(cè)定線粒體3態(tài)呼吸、線粒體呼吸控制率(RCR),與線粒體呼吸酶活性[細(xì)胞色素C氧化酶(Cc O)、NADH氧化酶(NADHO)及琥珀酸氧化酶(SUCO)]。透射電鏡觀察心肌細(xì)胞線粒體超微結(jié)構(gòu)。原代培養(yǎng)乳鼠心肌細(xì)胞,分別在低糖與高糖濃度下培養(yǎng)48h后建立缺氧/復(fù)氧損傷模型,實(shí)施SPost C(復(fù)氧開(kāi)始時(shí)吸入2.4%七氟醚)。缺氧或缺血前給予YC-1(終濃度為100μmol/L)、2ME2(2μmol/L)、環(huán)孢素A(終濃度0.2μmol/L)、Mdivi-1(終濃度50μmol/L或100μmol/L)進(jìn)行干預(yù),觀察心肌細(xì)胞復(fù)氧后的死亡率、LDH水平、細(xì)胞存活率,線粒體形態(tài)改變、mPTP開(kāi)放程度及線粒體膜電位變化。應(yīng)用western blotting或免疫熒光法測(cè)定線粒體融合分裂關(guān)鍵蛋白(Drp1、Fis1、Mfn1、Mfn2與Opa1)的表達(dá)水平,HIF-1α表達(dá)水平。結(jié)果:在體IRI模型中,SPost C通過(guò)增加JAK2/STAT3的磷酸化水平降低再灌注后心梗面積,保持線粒體完整結(jié)構(gòu)(P0.05,SPostC組vs I/R組)。離體灌流模型中,SPostC穩(wěn)定HIF-1α恢復(fù)線粒體呼吸功能與呼吸酶活性(P0.05,SPost C組vs I/R組)。細(xì)胞實(shí)驗(yàn)缺氧復(fù)氧損傷后,SPostC通過(guò)增加線粒體內(nèi)部連接度與拉長(zhǎng)度,抑制mPTP開(kāi)放、維持線粒體膜電位從而降低LDH水平與細(xì)胞死亡率,增加細(xì)胞存活率發(fā)揮心肌保護(hù)作用(P0.05,SPostC組vs H/R組)。但是HIF-1α抑制劑消除了這一保護(hù)作用(P0.05,YC-1組vs SPostC組)。高糖濃度下,CsA未能降低缺氧/復(fù)氧損傷后的LDH水平與細(xì)胞死亡率(P0.05),線粒體平均面積/周長(zhǎng)比與線粒體膜電位水平變化也無(wú)統(tǒng)計(jì)學(xué)差異(P0.05);但應(yīng)用Drp1抑制劑Mdivi-1恢復(fù)了高糖濃度下SPostC對(duì)線粒體形態(tài)的影響(P0.05),挽救了高糖狀態(tài)下SPost C對(duì)細(xì)胞死亡率、LDH水平與細(xì)胞存活率的作用(P0.05)。結(jié)論:SPostC通過(guò)激活大鼠JAK2-STAT3信號(hào)通路抵抗缺血/再灌注損傷發(fā)揮心肌保護(hù)作用。SPost C通過(guò)調(diào)節(jié)線粒體融合分裂關(guān)鍵蛋白Mfn2、Opa1、Drp1的表達(dá),增加線粒體連接度(平均面積/周長(zhǎng))與拉長(zhǎng)度(反圓度),維持線粒體融合分裂平衡,抑制mPTP通道開(kāi)放同時(shí)穩(wěn)定線粒體膜電位。HIF-1α是SPostC維持線粒體融合分裂平衡,改善心肌線粒體呼吸功能及呼吸酶活性發(fā)揮心肌保護(hù)作用的關(guān)鍵分子。體外細(xì)胞實(shí)驗(yàn),高糖降低線粒體連接性與拉長(zhǎng)度、促進(jìn)線粒體分裂致mPTP通道開(kāi)放和線粒體膜電位的降低,消除了SPostC的心肌保護(hù)效果,并且此作用與Drp1增高及Opa1表達(dá)不足有關(guān)。缺氧前應(yīng)用環(huán)孢素A抑制m PTP開(kāi)放無(wú)法恢復(fù)該保護(hù)效應(yīng),但應(yīng)用Drp1抑制劑Mdivi-1干預(yù)心肌細(xì)胞線粒體融合分裂過(guò)程,抑制高糖狀態(tài)下的線粒體過(guò)度分裂恢復(fù)了SPostC的心肌保護(hù)作用。
[Abstract]:Objective: cardiovascular disease is a serious threat to perioperative anesthesia and operation safety. Ischemia reperfusion injury (IRI) is a major cause of perioperative adverse cardiac events. Through the method of myocardial protection effect in the early stage of reperfusion of sevoflurane is known as sevoflurane (Sevoflurane Postconditionning, SPostC.SPostC) can produce similar ischemia hypoxia preconditioning for myocardial protection, which has been confirmed to occur for young healthy myocardial IRI has myocardial protective effect is very good, hope that the treatment of anti myocardial injury. However, the condition of diabetes, the effect of SPostC deletion or weakening of myocardial protection, seriously restricts the transformation and application of SPostC and protective effect. The use of insulin to the blood glucose levels in diabetic rats after myocardial recovery still cannot SPostC. How can the existing means of myocardial protection on the recovery of diabetes The protective effect of pathological myocardial, is an important clinical problem in the perioperative medicine needs to be solved. This research expects mitochondrial protection mechanism of sevoflurane by postprocessing, find the key protective effect of weakening, and observe the effect and effects of interference on sevoflurane postprocessing regulation of mitochondrial fusion division; finally through the regulation of mitochondrial fission and fusion balance recovery myocardial protective effect of SPostC under high glucose condition. Methods: establish the rat model of IRI and Langendorff from ischemia reperfusion heart model in rats. IRI model, 2h ischemia and 30min reperfusion, myocardial infarct size was determined, the contents of ATP and JAK2, the phosphorylation level of STAT3 from the Langendorff heart ischemia. At the end of reperfusion and reperfusion model, determination of mitochondrial state 3 respiration, mitochondrial respiratory control rate (RCR), oxidase and mitochondrial respiratory enzyme activity [cytochrome C (Cc O), NADH oxidase (NADHO) Succinate oxidase (SUCO) and observe the mitochondrial ultrastructure of myocardial cells. TEM. Primary cultured neonatal rat myocardial cells were cultured in 48h, low sugar and high glucose concentration after the hypoxia / reoxygenation injury model, the implementation of SPost C (inhalation of 2.4% sevoflurane reoxygenation started). Lack of oxygen or given before ischemia (YC-1 the final concentration of 100 mol/L), 2ME2 (2 mol/L), cyclosporin A (final concentration 0.2 mol/L), Mdivi-1 (final concentration of 50 mol/L or 100 mol/L) intervention, myocardial cells were observed after reoxygenation mortality, the level of LDH, the survival rate of cells, mitochondrial morphology change, change the opening degree and the mitochondrial membrane potential of mPTP. Determination of mitochondrial fission and fusion protein by Western or blotting key immunofluorescence (Drp1, Fis1, Mfn1, Mfn2 and Opa1) the expression level of HIF-1 alpha expression. Results: in the IRI model, SPost C by increasing the phosphorylation level of JAK2/STAT3 decreased again After reperfusion the myocardial infarction area, maintain complete mitochondrial structure (P0.05, SPostC group, vs group I/R). In vitro perfusion model, mitochondrial respiratory function and enzyme activity of SPostC alpha stable HIF-1 (P0.05, SPost group C recovery vs I/R group). Cells of experimental hypoxia reoxygenation injury after SPostC by increasing mitochondrial internal connectivity with the length of pull, inhibiting the opening of mPTP, maintaining mitochondrial membrane potential so as to reduce the level of LDH and the rate of cell death and increased cell survival rate play a role in myocardial protection (P0.05, SPostC group vs group H/R). But HIF-1 alpha inhibitors eliminates this protective effect (P0.05, YC-1 group vs group SPostC). Under high concentration of glucose, CsA to reduce the level of LDH cells to hypoxia / reoxygenation injury and mortality of oxygen (P0.05), average area / perimeter ratio changes of mitochondria and mitochondrial membrane potential level had no significant difference (P0.05); but the application of Drp1 inhibitor Mdivi-1 recovered under high concentration of glucose SPo Effect of stC on mitochondrial morphology (P0.05), to save the death rate of SPost cells C under high glucose condition, the level of LDH and the survival rate of cell function (P0.05). Conclusion: SPostC can activate the JAK2-STAT3 signaling pathway in rats against ischemia / reperfusion injury play a role in myocardial protection of.SPost C through the regulation of mitochondrial fission and fusion protein Mfn2. Opa1, Drp1 expression, increased mitochondrial connectivity (average area / perimeter) and length of pull (anti roundness), maintain the balance of mitochondrial fusion fission, inhibition of mPTP channel opening and the stability of mitochondrial membrane potential.HIF-1 SPostC alpha is to maintain mitochondrial fusion and fission balance of key molecules to improve mitochondrial respiratory function and respiratory enzyme activity in myocardium of myocardial preservation role. In vitro experiments, high glucose decreased mitochondrial connectivity and length of pull, reduce mitochondrial fission induced by mPTP channel opening and the mitochondrial membrane potential, elimination In addition to the myocardial protective effect of SPostC, and this effect with the increase of Drp1 and Opa1 expression insufficiency. Using cyclosporine A inhibits m PTP open not be able to restore the protective effect of hypoxia, but the application of mitochondrial Drp1 inhibitor Mdivi-1 on myocardial cell fusion fission process, inhibition of glucose under the situation of excess mitochondrial fission restored the myocardial protective effect of SPostC.

【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R614

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