本文選題：IRF-1 + JNK�。� 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:肝臟缺血再灌注(ischemia reperfusion,IR)損傷是臨床常見的病理過(guò)程,常發(fā)生在休克、創(chuàng)傷、肝切除和肝移植后,可導(dǎo)致術(shù)后肝功能恢復(fù)延遲甚至移植肝無(wú)功能,嚴(yán)重影響肝臟手術(shù)的臨床效果與患者生存預(yù)后。延長(zhǎng)肝缺血耐受時(shí)間、減少缺血再灌注損傷、保護(hù)移植肝功能是臨床亟待解決的重要問(wèn)題。本研究旨在探討小鼠肝臟缺血再灌注過(guò)程中,IRF-1表達(dá)對(duì)細(xì)胞自噬的影響,IRF-1調(diào)節(jié)JNK通路對(duì)肝缺血再灌注損傷自噬與損傷的作用,研究肝臟缺血再灌注損傷的可能機(jī)制,為肝臟缺血再灌注損傷的防治提供可能的方法。方法:雄性成年C57BL/6小鼠,隨機(jī)分為假手術(shù)組(Sham組),缺血再灌注0h、2h、6h、12h、24h組。采取夾閉肝左葉和中葉肝門制備70%肝缺血再灌注模型,檢測(cè)各組小鼠血清丙氨酸轉(zhuǎn)氨酶(ALT)、天冬氨酸轉(zhuǎn)氨酶(AST)水平。HE染色觀察肝臟組織病理學(xué)改變,TUNEL法觀察肝細(xì)胞凋亡情況。免疫組織化學(xué)檢測(cè)各組肝組織PCNA、Bcl-2和p-JNK的陽(yáng)性表達(dá),透射電鏡檢測(cè)小鼠肝組織細(xì)胞內(nèi)自噬體的數(shù)量,Western blot檢測(cè)小鼠肝組織IRF-1、JNK、p-JNK、Beclin-1、p62、LC3、Bcl-2、Caspase-3與Cleave Caspase-3蛋白表達(dá)變化。采用肝細(xì)胞系A(chǔ)ML12細(xì)胞建立缺氧/復(fù)氧(H/R)細(xì)胞模型以模擬肝缺血再灌注,進(jìn)一步驗(yàn)證體外實(shí)驗(yàn)IRF-1調(diào)控JNK誘導(dǎo)自噬性死亡在缺氧/復(fù)氧中的作用。通過(guò)Ad IRF-1、JNK si RNA預(yù)處理轉(zhuǎn)染AML12細(xì)胞,Western blot檢測(cè)各組AML12細(xì)胞IRF-1、JNK、p-JNK、Beclin-1、p62、LC3、Bcl-2、Caspase-3與Cleave Caspase-3蛋白表達(dá)變化,免疫細(xì)胞熒光檢測(cè)AML12細(xì)胞p-JNK的表達(dá)變化,共聚焦顯微鏡和透射電鏡觀察各組AML12細(xì)胞內(nèi)自噬體變化。使用雷帕霉素(Rap)與3-MA預(yù)處理AML12細(xì)胞,MTT檢測(cè)各組AML12細(xì)胞生存率,Western blot檢測(cè)各組AML12細(xì)胞內(nèi)Beclin-1、p62、LC3、Bcl-2、Caspase-3與Cleave Caspase-3蛋白表達(dá)變化,以明確自噬對(duì)H/R處理誘導(dǎo)AML12細(xì)胞凋亡的影響。結(jié)果:IR組小鼠血清ALT、AST水平隨著再灌注時(shí)間增加明顯上升(P0.05);IR組小鼠肝組織隨著再灌注時(shí)間增加損傷逐漸加重,肝組織細(xì)胞水腫、氣球樣變,肝竇區(qū)狹窄、中央靜脈淤血和肝細(xì)胞壞死等病理變化。與Sham組相比,IR組小鼠肝組織凋亡細(xì)胞隨著再灌注時(shí)間的增長(zhǎng)而明顯增多(P0.05)。與Sham組相比,IR組小鼠肝組織PCNA陽(yáng)性表達(dá)的細(xì)胞數(shù)隨著再灌注時(shí)間增加而升高,在6h達(dá)到高峰。IR組小鼠肝組織Bcl-2陽(yáng)性表達(dá)的細(xì)胞數(shù)隨著再灌注時(shí)間增加而減少,在12h表達(dá)最少。IR組小鼠肝組織p-JNK陽(yáng)性表達(dá)的細(xì)胞數(shù)在再灌注2h達(dá)峰值,然后隨再灌注時(shí)間增加而減少。與Sham組相比,再灌注6h組小鼠肝組織的自噬體的數(shù)量明顯升高。IR組小鼠肝組織IRF-1、JNK與p-JNK蛋白水平隨再灌注時(shí)間增加而升高,且均在2h達(dá)高峰,凋亡蛋白Cleave Caspase-3蛋白水平隨著再灌注時(shí)間增加而升高,在6h達(dá)高峰,Caspase-3蛋白水平?jīng)]有明顯變化,而抗凋亡蛋白Bcl-2蛋白水平減少。自噬相關(guān)蛋白Beclin-1、p62、LC3表達(dá)水平增加,在6h達(dá)高峰。Ad IRF-1組AML12細(xì)胞IRF-1、JNK與p-JNK蛋白水平較Ad GFP組升高。免疫熒光結(jié)果Ad IRF-1組AML12細(xì)胞p-JNK表達(dá)水平較Ad GFP組升高。相比Ad GFP組,Ad IRF-1組AML12細(xì)胞自噬相關(guān)蛋白Beclin-1、p62、LC3表達(dá)水平增加,Caspase-3與Cleave Caspase-3蛋白水平升高,而Bcl-2蛋白水平降低。相比si RNA-NC組,JNK si RNA組AML12細(xì)胞JNK和p-JNK表達(dá)減少,Beclin-1、p62、LC3、Caspase-3與Cleave Caspase-3水平降低,而Bcl-2蛋白水平升高。共聚焦和透射電鏡結(jié)果說(shuō)明Ad IRF-1組AML12細(xì)胞自噬體增加,而JNK si RNA組自噬體減少。使用自噬激動(dòng)劑和抑制劑預(yù)處理AML12細(xì)胞后,Rap組AML12細(xì)胞生存率較IR組明顯降低(P0.001),而3-MA組AML12細(xì)胞生存率較IR組明顯升高(P0.01)。Rap組Beclin-1、p62、LC3與Caspase-3與Cleave Caspase-3蛋白表達(dá)水平均較IR組顯著升高,而3-MA組較IR組明顯減少。結(jié)論:小鼠肝臟缺血再灌注誘導(dǎo)肝組織IRF-1與JNK信號(hào)通路表達(dá)增加,自噬水平升高,自噬體數(shù)量增加,自噬相關(guān)蛋白Beclin-1、p62、LC3的表達(dá)增高。同時(shí),凋亡相關(guān)蛋白Cleave Caspase-3蛋白表達(dá)增加。肝缺血再灌注過(guò)程中,IRF-1通過(guò)調(diào)控JNK信號(hào)通路增加肝細(xì)胞自噬水平,促進(jìn)肝細(xì)胞凋亡,加重缺血再灌注損傷。
[Abstract]:Objective: ischemia reperfusion (IR) injury is a common clinical pathological process, which often occurs after shock, trauma, hepatectomy and liver transplantation, which can lead to delayed recovery of liver function and no function of liver transplantation, which seriously affects the clinical effect of liver surgery and the survival prognosis of the patients. The purpose of this study is to explore the effect of IRF-1 expression on autophagy in the process of liver ischemia reperfusion in mice. IRF-1 regulates the role of JNK pathway in the autophagy and injury of liver ischemia-reperfusion injury, and studies the possible mechanism of liver ischemia reperfusion injury. Methods: the prevention and control of hepatic ischemia reperfusion injury was provided. Methods: male adult C57BL/6 mice were randomly divided into sham operation group (group Sham), ischemia reperfusion 0h, 2h, 6h, 12h, 24h group. A model of 70% liver ischemia reperfusion was made by clamping the left lobe of the liver and the middle lobe of the liver. The serum alanine transaminase (ALT) and aspartate aminotransferase (aspartate aminotransferase) were measured. (AST) the pathological changes of liver tissue were observed by the level of.HE staining, and the apoptosis of liver cells was observed by TUNEL method. The positive expression of PCNA, Bcl-2 and p-JNK in liver tissues of each group was detected by immunohistochemistry. The number of autophagic bodies in the liver tissues of mice was detected by transmission electron microscopy. Western blot detected the IRF-1, JNK, p-JNK, Beclin-1, Beclin-1. The changes in the expression of pase-3 and Cleave Caspase-3 protein. The hypoxia / reoxygenation (H/R) cell model was established by using hepatocyte line AML12 cells to simulate hepatic ischemia reperfusion. The effect of IRF-1 on the regulation of JNK induced autophagic death in hypoxia / reoxygenation in vitro was further verified. AML12 cells IRF-1, JNK, p-JNK, Beclin-1, p62, LC3, Bcl-2, Caspase-3 and Cleave Caspase-3 protein expression changes, immunofluorescence detection of the expression of AML12 cells, confocal microscopy and transmission electron microscopy to observe the changes in the autophagic cells in each group. Group AML12 cell survival rate and Western blot detection of Beclin-1, p62, LC3, Bcl-2, Caspase-3 and Cleave Caspase-3 protein expression in each group of AML12 cells to determine the effect of autophagy on the apoptosis induced by H/R treatment. With the time of reperfusion, the injury gradually increased, the hepatic tissue cell edema, the balloon like change, the stenosis of the hepatic sinus, the central venous congestion and the necrosis of the liver cells. Compared with the Sham group, the apoptotic cells of the liver tissue in the IR group increased significantly (P0.05). Compared with the group Sham, the PCNA positive form of the liver tissue in the group IR mice was compared with that of the group IR. The number of cells increased with the increase of reperfusion time. The number of Bcl-2 positive cells expressed in the liver tissue of the.IR group at the peak of 6h decreased with the increase of reperfusion time. The number of p-JNK positive cells in the liver tissue of the least.IR group in the.IR group was at the peak of the reperfusion 2h, and then decreased with the increase of the reperfusion time. And Sham group. In contrast, the number of autophagic bodies in the liver tissue of the 6h group was significantly increased in the liver tissue of.IR mice, and the level of JNK and p-JNK protein increased with the increase of reperfusion time, and at the peak of 2h, the level of the apoptotic protein Cleave Caspase-3 protein increased with the increase of reperfusion time, and the level of Caspase-3 protein was not obvious at the peak of 6h. The level of anti apoptotic protein Bcl-2 protein decreased. The expression level of autophagy related protein Beclin-1, p62 and LC3 increased, and the IRF-1, JNK and p-JNK protein levels of AML12 cells in the peak.Ad IRF-1 group were higher than those of the Ad group. The level of cellular autophagy related protein Beclin-1, p62, LC3 increased, and the level of Caspase-3 and Cleave Caspase-3 increased, while the level of Bcl-2 protein decreased. The results of electron microscopy showed that the autophagic body of AML12 cells in the Ad IRF-1 group was increased, but the autophagic body of the JNK Si RNA group decreased. The survival rate of AML12 cell in Rap group was significantly lower than that of the IR group after the pretreatment of AML12 cells with autophagic agonists and inhibitors, while the survival rate of 3-MA group was more than that of Xian Shenggao. The expression level of leave Caspase-3 protein was significantly higher than that in the IR group, while the 3-MA group was significantly lower than the IR group. Conclusion: the expression of IRF-1 and JNK signaling in liver tissues of mice was increased, the level of autophagy increased, the number of autophagic increased, the expression of autophagic related protein Beclin-1, p62 and LC3 increased. Meanwhile, the apoptosis related protein was Cleave Cas The expression of pase-3 protein is increased. In the course of liver ischemia and reperfusion, IRF-1 increases the autophagy level of hepatocyte by regulating the JNK signaling pathway, promotes the apoptosis of liver cells and aggravates the ischemia reperfusion injury.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R657.3

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