本文選題：七氟醚 + 后處理��； 參考：《蘇州大學》2015年博士論文

【摘要】：第一部分七氟醚后處理減輕離體大鼠心肌缺血再灌注損傷目的通過對比心肌I/R前后血液動力學參數(shù)、凋亡和梗死面積,探討七氟醚后處理對大鼠心肌缺血再灌注損傷的保護作用。方法建立大鼠心臟Langendorff離體心臟灌流模型,取制備離體模型成功的心臟32個,分為4組(n=8):假手術(shù)組(SHAM組),單純七氟醚組(S組),單純?nèi)毖俟嘧⒔M(I/R組),七氟醚后處理組(SEVOP組)。(1)SHAM組:K-H液持續(xù)灌注180 min;(2)S組:K-H液持續(xù)灌注60 min,且于60min時給予含2.5%七氟醚(批號:2217,丸石制藥株氏會社,日本)的K-H液灌注15 min,繼續(xù)K-H液灌注105 min;(3)I/R組:平衡30 min,缺血30 min,再灌注120 min;(4)SEV0P組:于再灌注初給予含2.5%七氟醚的K-H液灌注15 min;記錄各組血液動力學參數(shù);TUNEL法檢測細胞凋亡部分;TTC法計算心肌梗死面積;Western blot測Bcl-2、Caspase-3的蛋白表達。結(jié)果與T0比,I/R和SEVOP組在T1、T2、T3、T4時點LVSP、HR、±dp/dtmax、降低(P0.05),LVEDP升高(P0.05);在T1、T2、T3、T4時刻,與SHAM組比較,I/R和SEVOP組LVSP、HR、±dp/dtma降低,LVEDP升高(P0.05);與I/R組比較,SEVOP組LVSP、HR、±dp/dtma升高,LVEDP降低(P0.05)。與I/R組(51±5)%相比,SEVOP組(31±4)%的心肌梗死范圍縮小(P0.05)。TUNEL法染色結(jié)果示,與SHAM組相比,I/R組及SEVOP組心肌細胞TUNEL染色陽性細胞增多,TUNEL凋亡指數(shù)增高(P0.05);與I/R組比,SEVOP組心肌細胞TUNEL染色陽性細胞較少,凋亡指數(shù)較低(P0.05)。與SHAM組相比,I/R組及SEVOP組心肌Bcl-2蛋白表達下降,Caspase-3表達增加,差別有統(tǒng)計學意義。(P0.05);與I/R組比,SEVOP組心肌Bcl-2蛋白表達升高,Caspase-3表達下降,差別有統(tǒng)計學意義。(P0.05)結(jié)論七氟醚后處理后大鼠左室舒張功能增加,心肌收縮力增強,心功能改善明顯;七氟醚后處理能抑制心肌細胞凋亡,減小梗死面積,減輕大鼠心肌缺血再灌注損傷。第二部分七氟醚后處理減輕離體大鼠心肌缺血再灌注損傷的分子機制:NOS/NO-NHE1-MPTP途徑目的通過心肌I/R后,七氟醚后處理,用或不用L-NAME后NOS/NO、NHE1/p-NHE1、和NAD+含量變化,探討七氟醚后處理離體大鼠心肌缺血再灌注損傷的保護作用的分子機制。方法Langendorff灌流模型成功的雄性SD大鼠離體心臟72個,采用隨機數(shù)字表法,將其分為6組(n=12):假手術(shù)組(S組):連續(xù)用K-H液灌注180 min;七氟醚組(Sev組):K-H液灌注60 min后,用含2.5%七氟醚的K-H液灌注15 min,再用K-H液灌注105 min;缺血再灌注組(I/R組):K-H液灌注30 min后,停灌30 min,再灌注120min,制備心肌缺血再灌注損傷模型;七氟醚后處理組(SEVOP組):K-H液灌注30 min后,停灌30 min,于再灌注開始即刻用含2.5%七氟醚的K-H液灌注15 min,隨后再用K-H液灌注105 min;七氟醚后處理+NOS抑制劑旋硝基精氨酸甲基酯(L-NAME)組(SEVOP+L-NAME組):再灌注開始用含濃度為100μmol/L L-NAME的K-H液灌注60 min(再灌注開始同時注入K-H液2.5%七氟醚并維持15分鐘),隨后再K-H液灌注60 min;NOS抑制劑旋硝基精氨酸甲基酯(L-NAME)組(L組):再灌注開始即刻用含濃度為100μmol/L L-NAME的K-H液灌注60 min,再換用上述單純K-H液灌注60 min;于再灌注2 h時取心肌組織,采用分光光度法檢測NO、NAD+含量和NOS活性,Western Blot法測定總NHE1(t-NHE1)和磷酸化NHE1(p-NHE1)的表達,RT-PCR法測定NHE1 m RNA的表達水平,并測定心肌梗死體積,觀察心肌超微結(jié)構(gòu)。結(jié)果與S組比較,I/R組、SEVOP組、SEVOP+L-NAME組和L組心肌梗死體積增大,心肌組織NO、NOS和NAD+水平降低,p-NHE1及NHE1 m RNA表達上調(diào)(P0.05),Sev組上述指標差異無統(tǒng)計學意義(P0.05);與I/R組比較,SEVOP組心肌梗死體積減小,心肌組織NO、NOS和NAD+水平升高,p-NHE1及NHE1 m RNA表達下調(diào)(P0.05),SEVOP+L-NAME組和L組上述指標差異無統(tǒng)計學意義(P0.05);與SEVOP組比較,SEVOP+L-NAME組和L組心肌梗死體積增大,心肌組織NO、NOS和NAD+水平降低,p-NHE1及NHE1 m RNA表達上調(diào)(P0.05)。SEVOP組心肌病理學損傷較I/R組和SEVOP+L-NAME組減輕。結(jié)論七氟醚后處理可能通過增強心肌NOS活性,促進NO合成,抑制NHE1功能,減少MPTP開放,從而減輕大鼠離體心臟缺血再灌注損傷。第三部分七氟醚后處理對離體大鼠缺血再灌注心肌脹亡和自噬的影響目的觀察七氟醚后處理對大鼠心肌脹亡和自噬活動的影響。方法建立大鼠心臟Langendorff離體心臟灌流模型,取制備離體模型成功的心臟32個,分為4組(n=8):假手術(shù)組(SHAM組),單純七氟醚組(S組),單純?nèi)毖俟嘧⒔M(I/R組),七氟醚后處理組(SEVOP組),各組處理方法同第一部分。透射電鏡觀察細胞脹亡和自噬;Western blot測自噬相關(guān)分子LC3I、LC3II,Beclin-1和脹亡相關(guān)分子porimin的蛋白表達。結(jié)果Sham組自噬小體數(shù)目是1.2±0.7,脹亡細胞數(shù)是3.2±1.5;S組自噬小體的數(shù)目是1.0±0.6,脹亡細胞數(shù)是3.0±1.3,兩組對比,無統(tǒng)計學差異。而在缺血再灌注組,I/R組自噬小體數(shù)目是10.4±2.4,脹亡細胞數(shù)是15.5±4.2;七氟醚后處理組SEVOP組自噬小體的數(shù)目是6.8±1.8,脹亡細胞數(shù)是11.7±3.5表明缺血再灌注后自噬小體和脹亡細胞數(shù)目顯著增加,而七氟醚后處理能顯著減少噬小體和脹亡細胞生成的數(shù)目。結(jié)論單獨使用七氟醚不影響自噬小體和脹亡細胞數(shù)目,缺血再灌注后自噬小體和脹亡細胞數(shù)目顯著增加,而七氟醚后處理能顯著減少自噬小體和脹亡細胞生成的數(shù)目。
[Abstract]:The first part of sevoflurane relieves myocardial ischemia and reperfusion injury in isolated rat myocardium by comparing the hemodynamic parameters, apoptosis and infarct area before and after I/R, and exploring the protective effect of sevoflurane postconditioning on myocardial ischemia reperfusion injury in rats. Methods to establish a rat heart perfusion model of cardiac Langendorff in vitro, and prepare the preparation of the rat heart perfusion model. 32 successful hearts in the isolated model were divided into 4 groups (n=8): sham operation group (group SHAM), simple sevoflurane group (group S), simple ischemia reperfusion group (group I/R), sevoflurane group (group SEVOP). (1) SHAM group: K-H solution continuous perfusion 180 min; (2) S group: K-H liquid continuous perfusion 60 min, and 2.5% sevoflurane in 60min (batch number: 2217, pill stone pharmaceutical strain) The K-H solution in the society, Japan, was perfused 15 min, continued the K-H solution of 105 min, and (3) I/R group: balance 30 min, ischemia 30 min, and reperfusion 120 min; (4) SEV0P group: 2.5% sevoflurane containing K-H solution was given at first, and the hemodynamic parameters of each group were recorded, the apoptotic part of the cells was detected by TUNEL method. Bcl-2, Caspase-3 protein expression. Results compared with T0, I/R and SEVOP group in T1, T2, T3, T4 time point LVSP, HR, dp/dtmax, decreased. Compared with group R (51 + 5)%, SEVOP group (31 + 4)% of myocardial infarct scope reduced (P0.05).TUNEL staining results, compared with group SHAM, TUNEL staining positive cells in group I/R and SEVOP group increased and TUNEL apoptosis index increased (P0.05), and the number of apoptotic cells in SEVOP group was less and the apoptosis index was lower than that in I/R group. Compared with group I/R and group SEVOP, the expression of Bcl-2 protein decreased and the expression of Caspase-3 increased. (P0.05). Compared with group I/R, the expression of Bcl-2 protein in SEVOP group increased and Caspase-3 expression decreased, and the difference was statistically significant. (P0.05) conclusion the left ventricular diastolic function of rats after sevoflurane after treatment was increased and the contractility of myocardium was enhanced. The treatment of sevoflurane can inhibit cardiomyocyte apoptosis, reduce infarct size and reduce myocardial ischemia reperfusion injury in rats. The second part of sevoflurane relieves the molecular mechanism of myocardial ischemia reperfusion injury in isolated rat myocardium: after the NOS/NO-NHE1-MPTP pathway, after I/R, sevoflurane is treated with or without L-N After AME NOS/NO, NHE1/p-NHE1, and NAD+ content changes, the protective effects of sevoflurane on myocardial ischemia and reperfusion injury in isolated rat myocardium were investigated. Methods 72 isolated male SD rats were successfully treated with Langendorff perfusion model, and they were divided into 6 groups (n=12): the sham operation group (S group): perfusion of K-H solution of 18. 0 min, sevoflurane group (group Sev): after K-H solution was perfused 60 min, 15 min was perfused with K-H solution containing 2.5% sevoflurane, and 105 min was perfused with K-H solution; ischemia reperfusion group (I/R group): after K-H solution was perfused 30 min, 30 min was stopped and reperfusion injury model of myocardial ischemia and reperfusion was prepared. N, infusion of 15 min with 2.5% sevoflurane solution at the beginning of reperfusion, followed by 105 min with K-H solution, and after sevoflurane treatment of +NOS inhibitor, +NOS inhibitor, group SEVOP+L-NAME (SEVOP+L-NAME group): reperfusion began to pour 60 min (2.5% seven fluorine at the beginning of reperfusion) with a K-H liquid containing a concentration of 100 micron L-NAME. The ether was maintained for 15 minutes, and then the K-H solution was followed by 60 min, and the NOS inhibitor, the group of L-NAME (group L), was injected with the K-H solution containing 100 mu mol/L L-NAME, and then perfused 60 min, and then changed to 60 min by the pure K-H solution. NOS activity, Western Blot method was used to determine the expression of total NHE1 (t-NHE1) and phosphorylated NHE1 (p-NHE1). RT-PCR assay was used to determine the expression level of NHE1 m RNA, and the volume of myocardial infarction was measured and the myocardial ultrastructure was observed. The expression of E1 and NHE1 m RNA was up-regulated (P0.05), and there was no significant difference in the above indexes in group Sev (P0.05). Compared with group I/R, the volume of myocardial infarction decreased in SEVOP group, NO, NOS and NAD+ levels in the myocardium. Myocardial infarction volume in group P+L-NAME and group L increased, myocardial tissue NO, NOS and NAD+ decreased, p-NHE1 and NHE1 m RNA expression was up regulation (P0.05).SEVOP group. The effect of third part of sevoflurane on myocardial atolis and autophagy in rats in vitro. Objective To observe the effect of sevoflurane on cardiac atolum and autophagy in rats. Methods a rat heart perfusion model of Langendorff in vitro was established. 32 hearts were divided into 4 groups (n=8): sham operation group (group SHAM), simple sevoflurane group (group S), simple ischemia-reperfusion group (group I/R), and sevoflurane group (group SEVOP). The treatment methods of each group were the same as the first part. Transmission electron microscopy was used to observe the cell atoly and autophagy; Western blot was used to detect autophagic related molecules LC3I, LC3II, Beclin-1, and bulging associated molecules. Results the number of autophagic corpuscles in group Sham was 1.2 + 0.7, the number of bulging cells was 3.2 + 1.5, the number of autophagic bodies in group S was 1 + 0.6, the number of bulging cells was 3 + 1.3, and there was no statistical difference between the two groups. In the ischemia reperfusion group, the number of autophagic corpuscles in the group I/R was 10.4 + 2.4, the number of the bulging cells was 15.5 + 4.2, and the SEVOP group of the sevoflurane post treatment group. The number of autophagic bodies was 6.8 + 1.8, and the number of bulging cells was 11.7 + 3.5. The number of autophagic corpuscles and bulging cells increased significantly after ischemia-reperfusion, while sevoflurane reprocessing could significantly reduce the number of phagocytic and bulging cells. The number of phagocytic bodies and apoptotic cells increased significantly, while sevoflurane postconditioning significantly reduced the number of autophagic bodies and the number of apoptotic cells.
【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R614

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