發(fā)布時間：2018-04-06 20:02

  本文選題：膿毒癥　切入點：硫氫化鈉　出處：《南方醫(yī)科大學》2017年博士論文

【摘要】：目的:選擇最合適盲腸結扎部位行盲腸結扎穿刺法(cecal ligation and puncture,CLP),建立本實驗最適膿毒癥模型,首次探討膿毒癥對小鼠心肌線粒體生物合成的影響;給予不同濃度硫氫化鈉(sodium hydrosulfide,NaHS)干預,首次探討NaHS對膿毒癥小鼠心肌線粒體的保護作用、最佳濃度及相關機制,為膿毒癥動物實驗研究及臨床診治提供理論基礎。方法:1.取40只C57BL/6小鼠隨機分為假手術組(Sham)和CLP1、CLP2、CLP3組(分別結扎盲腸遠端1/4、1/2、3/4),比較小鼠生存情況,選擇最佳模型和取材時間點。2.另取50只C57BL/6小鼠隨機分為CLP、CLP + NaHS(4亞組,NaHS濃度分別為25、50、100、2000μmol/L)5組,比較小鼠生存情況。3.另取70只C57BL/6小鼠隨機分為正常對照組(Control)、Sham、CLP、CLP+NaHS(4亞組,NaHS濃度同前)7組,檢測和比較以下指標:(1)小鼠膿毒癥表現(xiàn)。(2)血漿內毒素水平(3)心肌損傷:HE染色觀察心肌細胞形態(tài)、ELISA檢測血清肌鈣蛋白(cardiac troponin I,cTnI)水平。(4)心肌線粒體損傷:透射電鏡觀察線粒體形態(tài)學,分光光度法檢測線粒體腫脹程度,生物發(fā)光法檢測ATP水平。(5)氧化應激:TBA 法檢測丙二醛(malondialdehyde,MDA)水平,DCFH-DA熒光探針法檢測活性氧(reactive oxygen species,ROS)水平。(6)抗氧化物:比色定量法檢測谷胱甘肽(glutathione,GSH)水平;Westem Blot法檢測錳超氧化物歧化酶(Mn-superoxide dismutase,MnSOD)、血紅素加氧酶 1(heme oxygenase-1,HO-1)蛋白水平;RT-qPCR 法檢測 MnSOD、HO-1 mRNA水平。(7)線粒體生物合成相關因子:RT-qPCR法檢測過氧化物酶體增殖物激活受體 γ 輔激活子 1α(peroxisome proliferator-activated receptor-γ coactivator-1α,PGC-1α)、核因子 E2 相關因子 2(nuclear factor-erythroid-2-related factor 2,Nrf2)、線粒體轉錄因子 A(mitochondrial transcription factor A,Tfam)mRNA 水平。結果:1.CLP1、CLP2、CLP3組小鼠建模后開始死亡時間分別是12h、11h、8h,CLP1組小鼠48h存活率高于CLP2、CLP3組,故選擇CLP1為本研究實驗模型,建模后12h為取材時間點。2.NaHS干預組小鼠48h存活率高于CLP組,100μmol/L組最高。3.CLP組小鼠建模后6小時出現(xiàn)膿毒癥表現(xiàn),逐漸加重;NaHS干預組膿毒癥表現(xiàn)減輕,NaHS濃度越高,癥狀越輕。4.CLP組內毒素水平升高。5.CLP組可見HE染色心肌細胞形態(tài)異常、cTnI水平升高;NaHS干預組心肌細胞形態(tài)異常減輕、cTnI水平下降,NaHS濃度越高作用越明顯。6.CLP組心肌線粒體形態(tài)異常、腫脹程度增加、ATP水平下降;NaHS干預后線粒體形態(tài)異常及腫脹程度減輕,ATP水平升高,NaHS濃度越高作用越明顯。7.CLP組ROS、MDA水平升高;與CLP組比較,各濃度NaHS組MDA水平均下降,但100、2000μmol/L組間無顯著性差異,ROS水平僅100、200μmol/L組下降。8.CLP組GSH水平下降、MnSOD、HO-1蛋白及mRNA水平上升;NaHS干預組其水平均較CLP組上升,且呈NaHS濃度依賴性,200μmol/L組MnSOD、HO-1蛋白水平低于100μmol/L組。9.CLP 組 PGC-1α、Nrf2、Tfam mRNA 水平均上升;NaHS 干預組較 CLP組繼續(xù)上升,NaHS濃度越高,上升越明顯。10.CLP組和NaHS干預組cTnI水平、線粒體腫脹程度均與ROS、MDA水平呈正相關,ATP水平與ROS、MDA水平呈負相關;NaHS干預組ROS、MDA水平與GSH水平、MnSOD及HO-1 mRNA水平呈負相關,ATP、GSH水平及MnSOD、HO-1、Tfam mRNA 水平均與 PGC-1α、Nrf2 mRNA 水平呈正相關。結論:1.膿毒癥可造成心肌線粒體氧化應激損傷。2.NaHS呈濃度依賴性減輕膿毒癥時心肌線粒體損傷,100μmol/L為最佳濃度。3.NaHS可通過PGC-1α、Nrf2上調膿毒癥時抗氧化物水平、促進線粒體生物合成,從而保護心肌線粒體。
[Abstract]:Objective: to choose the most suitable site for cecal ligation and cecal ligation and puncture method (cecal ligation and puncture, CLP), to establish the optimal model of sepsis in this study, for the first time to investigate the effect of sepsis on myocardial mitochondrial biogenesis in mice; with different concentrations of sodium hydrosulfide (sodium hydrosulfide, NaHS) for the first time to investigate the protective effect of NaHS intervention. In sepsis mice myocardial mitochondria, the optimal concentration and related mechanism, provide a theoretical basis for the study of animal experiment and clinical diagnosis and treatment of sepsis. Methods: 1. 40 C57BL/6 mice were randomly divided into sham operation group (Sham) and CLP1 group (CLP3, CLP2, 1/4,1/2,3/4 were ligated distal caecum), mice survival choose the best model, and at each time point.2. the other 50 C57BL/6 mice were randomly divided into CLP, CLP + NaHS (4 sub group, NaHS concentration was 25,501002000 ~ mol/L) 5 groups, mice survival.3. another 70 C5 7BL/6 mice were randomly divided into normal control group (Control), Sham, CLP, CLP+NaHS (4 sub group, the concentration of NaHS was 7) group to examine and compare the following indicators: (1) in mice with sepsis. (2) the level of plasma endotoxin (3) myocardial injury: Observation of myocardial cell morphology by HE staining, detection serum cardiac troponin ELISA (cardiac troponin I, cTnI) level. (4) myocardial mitochondria: To observe mitochondrial morphology by transmission electron microscopy, spectrophotometric detection of mitochondrial swelling, detect the level of ATP bioluminescence method. (5) oxidative stress: detection of C TBA two (malondialdehyde, MDA) level of formaldehyde, detection of reactive oxygen species DCFH-DA fluorescence probe method (reactive oxygen species, ROS) level. (6) the antioxidant glutathione detection: more than color quantitative method (glutathione, GSH); Westem Blot method for detection of manganese superoxide dismutase (Mn-superoxide dismutase, MnSOD), heme oxygenase 1 (heme oxygenase-1, HO-1 ) protein level; detection of MnSOD RT-qPCR, HO-1 mRNA. (7) factors related to mitochondrial biogenesis: detection of peroxisome proliferator activated receptor gamma RT-qPCR coactivator 1 alpha (peroxisome proliferator-activated receptor- coactivator-1 PGC-1 gamma alpha, alpha), nuclear factor E2 related factor 2 (nuclear factor-erythroid-2-related 2 factor, Nrf2), mitochondrial transcription factor A (mitochondrial transcription factor A, Tfam mRNA). Results: 1.CLP1, CLP2, CLP3 group of mice after death started modeling time were 12h, 11h, 8h, CLP1 group of mice survival rate of 48h is higher than CLP2, CLP3 group, the choice of CLP1 as the experimental model, after modeling 12h at each time point.2.NaHS the intervention group mice survival rate of 48h is higher than that of CLP group, sepsis, 6 hours of 100 mol/L were the highest in.3.CLP group after modeling gradually increased; NaHS intervention group sepsis reduction Light, the higher the concentration of NaHS, the more mild symptoms endotoxin level increased in.4.CLP group.5.CLP group showed HE staining abnormal myocardial cell morphology, elevated levels of cTnI; NaHS group of abnormal myocardial cell morphology intervention decreased, cTnI decreased, the higher the concentration of NaHS is more obvious in.6.CLP group myocardial mitochondrial abnormal morphology, swelling degree increased, the level of ATP after the intervention of NaHS decreased; abnormal mitochondrial morphology and reduce swelling, increased the level of ATP, the higher the concentration of NaHS is more obvious in.7.CLP group ROS, MDA level increased; compared with group CLP, the concentration of NaHS group MDA level decreased, but 1002000 mol/L had no significant difference, ROS level is only 100200 ~ mol/L group the level of GSH decreased in.8.CLP group decreased, MnSOD, HO-1 and mRNA protein level increased; the level of NaHS in the intervention group were lower than CLP group increased, and showed NaHS concentration dependent, 200 mol/L MnSOD group, a low level of HO-1 protein in 100 mol/L group.9.CLP group PGC -1 alpha, Nrf2, Tfam and mRNA levels were increased; the NaHS intervention group compared with the CLP Group continues to rise, the higher the concentration of NaHS increased more obviously,.10.CLP group and NaHS group the level of cTnI, the swelling of mitochondria and ROS, MDA level was positively correlated with ATP level and ROS level of MDA was negatively correlated; NaHS intervention group ROS, MDA level and GSH level was negatively correlated with MnSOD and HO-1 mRNA ATP, HO-1 GSH level and MnSOD, Tfam, mRNA and PGC-1 levels were positively related to the level of mRNA alpha, Nrf2. Conclusion: 1. sepsis can cause myocardial mitochondrial oxidative stress damage in.2.NaHS in a concentration dependent manner to reduce damage of myocardial mitochondria in sepsis when 100 mol/L was the best concentration of.3.NaHS by PGC-1 alpha, Nrf2 upregulated the antioxidant levels in sepsis, promote mitochondrial biogenesis, thereby protecting myocardial mitochondria.

【學位授予單位】：南方醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R459.7

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