本文選題：TXNIP + 心肌缺血/再灌注�。� 參考：《第四軍醫(yī)大學》2015年博士論文

【摘要】：研究背景心肌缺血后恢復血流可顯著減輕心肌損傷。然而血管的開通卻會發(fā)生缺血再灌注損傷1,2。進一步的研究表明,心肌細胞的自噬在缺血再灌注損傷中起到重要作用。正常情況下,心臟維持著低水平的自噬。心肌缺血時自噬升高,具有對抗缺血的保護作用。然而在再灌注期,本應下降的自噬卻繼續(xù)升高,損害了心臟功能并促進了心肌細胞損傷和死亡3。缺血再灌注時血流既已恢復,可自噬因何會繼續(xù)激活升高并損害心肌,其機制我們仍不清楚。TXNIP,又稱Trx相互結合蛋白4-6,在心肌缺血時表達升高。TXNIP敲除小鼠心肌缺血再灌注時,由于TXNIP的敲除導致細胞的能量代謝更傾向于無氧糖酵解,避免缺血所導致的線粒體進行的無效的氧化磷酸化,生成了更多的ATP7。既往報道,當細胞缺乏能量時,AMPK激活導致自噬8。既然TXNIP能夠調控心肌細胞再灌注時期的能量代謝,且又在此時表達過度升高,TXNIP是否是心肌缺血再灌注時調控自噬的分子呢,我們仍不清楚。因此,在后續(xù)實驗中我們圍繞此進行了系列實驗,以期證明缺血再灌注時TXNIP增加參與調控了自噬水平的提高。研究目的1.觀察過表達或敲除TXNIP是否影響MI/R后心肌損傷2.研究過表達或敲除TXNIP是否會影響心肌自噬水平3.闡述TXNIP調控自噬的具體信號通路研究方法1觀察過表達或敲除TXNIP是否影響MI/R后心肌損傷1.1構建并鑒定TXNIPfloxed/floxed Myosin6-Cre小鼠1.2通過心肌點注射腺病毒構建過表達TXNIP的小鼠1.3制備小鼠心肌缺血及心肌缺血再灌注模型用6-0絲線結扎小鼠左冠狀動脈后,將絲線打活結造成心肌缺血,缺血40分鐘后打開活結。分別在缺血40分鐘后、再灌注1.5小時及再灌注3小時后檢測心肌蛋白含量。在缺血40分鐘再灌注3小時后檢測心肌細胞凋亡,在再灌注24小時后檢測心功能及心肌梗死面積。假手術組采用同樣的手術方法,但并不結扎小鼠冠狀動脈1.4通過Western blot檢測心肌細胞TXNIP表達1.5通過小動物超聲檢測及左心室內插管測定小鼠小鼠心臟功能1.6通過伊文氏藍/TTC雙染法檢測小鼠心肌缺血再灌注后心肌梗死面積1.7通過TUNEL染色測定小鼠心肌細胞凋亡2研究過表達或敲除TXNIP是否會影響心肌自噬水平2.1鑒定LC3-GFP小鼠,同時構建TXNIPMyosin6-Cre-LC3-GFP小鼠及LC3-GFP-TXNIP過表達小鼠2.2制備小鼠心肌缺血及心肌缺血再灌注模型方法同前。分別在缺血40分鐘后及再灌注3小時后檢測心肌蛋白含量。在缺血40分鐘再灌注3小時后使用電鏡及熒光顯微鏡檢測小鼠自噬情況2.3通過Western blot檢測心肌組織LC3及P62的表達2.4使用透射電鏡觀察心肌自噬小體2.5使用熒光顯微鏡觀察小鼠LC3-GFP自噬小體3闡述TXNIP調控自噬的具體信號通路3.1鑒定AMPKfloxed/floxedMyosin6-Cre小鼠,構建AMPKfloxed/floxedMyosin6-Cre-TXNIP過表達小鼠3.2制備小鼠心肌缺血及心肌缺血再灌注模型方法同前。分別在缺血40分鐘后及再灌注3小時后檢測心肌蛋白含量及ATP含量。在缺血40分鐘再灌注3小時后使用電鏡檢測小鼠自噬情況3.3通過熒光法測定心肌組織ATP含量3.4通過Western blot檢測心肌細胞p AMPK、AMPK、p Raptor、Raptor、p ULK1、ULK1的表達3.5通過透射電鏡觀察相關小鼠心肌自噬小體3.6通過Western blot檢測心肌組織LC3表達研究結果1缺血所致的TXNIP升高加重心肌缺血再灌注損傷1.1心肌缺血及再灌注損傷過程中TXNIP持續(xù)升高1.2 TXNIP過表達加重了小鼠心肌缺血再灌注后的心臟功能降低TXNIP過表達小鼠顯著加重了心肌缺血再灌注損傷導致的左室射血分數(shù)降低。缺血再灌后,TXNIP過表達小鼠的LVEDP,+dp/dtmax和-dp/dtmax三個指標均明顯差于WT小鼠與TXNIP敲除小鼠。而TXNIP敲除小鼠的上述指標相比野生小鼠出現(xiàn)進一步的好轉1.3 TXNIP過表達增加了缺血再灌注后的心肌梗死面積及細胞凋亡與野生型小鼠相比,TXNIP過表達小鼠顯著增加心肌梗死面積及心肌細胞凋亡,而TXNIP敲除小鼠缺血再灌注損傷則顯著更輕2升高的TXNIP加強了心肌缺血再灌所導致的自噬增加2.1 TXNIP影響了缺血再灌注后心肌自噬相關蛋白的含量在缺血再灌注后,TXNIP過表達小鼠的LC3 II/LC3 I的比值進一步升高,與野生型小鼠和TXNIP敲除鼠相比均出現(xiàn)統(tǒng)計學差異。野生型小鼠與TXNIP敲除鼠的LC3 II/LC3 I的比值亦出現(xiàn)統(tǒng)計學差異,敲除鼠的比值顯著更低2.2 TXNIP影響了缺血再灌注后心肌細胞的自噬小體數(shù)量通過透射電鏡及觀察LC3-GFP小鼠的心肌細胞熒光斑點,我們發(fā)現(xiàn)自噬小體水平在TXNIP敲除鼠中顯著降低,而在TXNIP過表達小鼠中顯著升高3 TXNIP通過AMPK調控心肌缺血再灌導致的自噬增加3.1 TXNIP影響心肌缺血再灌注后ATP生成與野生型小鼠相比較,TXNIP敲除鼠的ATP含量在缺血再灌注后顯著更高,具有統(tǒng)計學差異,而TXNIP過表達小鼠的心肌ATP含量出現(xiàn)了顯著的下降3.2 TXNIP通過AMPK調節(jié)自噬在TXNIP過表達小鼠中,缺血再灌注導致AMPK、Raptor的磷酸化激活增加,而TXNIP敲除小鼠則可減少AMPK、Raptor的磷酸化激活,增加ULK1的磷酸化失活。進一步,在AMPK被敲除后,與具有AMPK活性的野生型小鼠相比,TXNIP過表達所導致的自噬小體數(shù)量顯著減少研究結論本課題證實了:1.TXNIP在缺血再灌注后的升高,且這種升高導致心臟功能的降低,心肌梗死面積的增加及細胞凋亡的增多。而敲除TXNIP則具有上述相反得作用。2.TXNIP并不影響心肌缺血期的自噬,而卻在再灌注期的升高增加了自噬,且其機制是通過AMPK所介導的。這些結果為我們進一步認識心肌缺血再灌注導致的自噬升高提供了新的思路。
[Abstract]:The recovery of blood flow after myocardial ischemia in the background can significantly reduce myocardial damage. However, the opening of the blood vessels can cause ischemic reperfusion injury 1,2. further research shows that the autophagy plays an important role in the ischemia reperfusion injury. There is a protective effect against ischemia. However, during the reperfusion period, the autophagy should continue to increase, damage the heart function and promote myocardial damage and death 3. ischemia reperfusion, the blood flow has been restored, autophagy can continue to activate and damage the myocardium, the mechanism of which we still do not know.TXNIP, also called Trx to combine eggs with each other. White 4-6, during ischemia and reperfusion in.TXNIP knockout mice during myocardial ischemia, the energy metabolism of cells is more inclined to anaerobic glycolysis due to the knockout of TXNIP, which avoids the ineffective oxidative phosphorylation of mitochondria caused by ischemia, resulting in more ATP7. previous reports that AMPK activation leads to the activation of cells when the cells are deficient in energy. Autophagy 8., since TXNIP can regulate the energy metabolism during the reperfusion period of cardiac myocytes, and is overexpressed at this time, we are still not clear about whether TXNIP is a molecule that regulates autophagy during myocardial ischemia and reperfusion. Therefore, we have carried out a series of experiments around this in a follow-up experiment to demonstrate the increase of TXNIP participation in ischemia reperfusion. Regulation of the improvement of autophagy level. 1. to observe whether the expression or knockout of TXNIP affects myocardial injury after MI/R research is 2. or whether the knockout of TXNIP affects myocardial autophagy level 3. and the specific signal pathway study method for TXNIP regulation of autophagy: 1 Observation of overexpression or knockout TXNIP affects 1.1 construction of myocardial injury after MI/R The TXNIPfloxed/floxed Myosin6-Cre mice 1.2 was constructed by injecting the adenovirus into the myocardium to construct TXNIP in mice 1.3 to prepare the myocardial ischemia and myocardial ischemia reperfusion model in mice. After ligating the left coronary artery with 6-0 silk lines, the silk thread was used to cause myocardial ischemia, and the ischemia was opened for 40 minutes after ischemia. 40 minutes after ischemia, the mice were formed. Myocardial protein content was detected after reperfusion for 1.5 hours and reperfusion for 3 hours. Cardiomyocyte apoptosis was detected after 3 hours of reperfusion after 40 minutes of ischemia. Cardiac function and myocardial infarction area were detected after 24 hours of reperfusion. The same operative method was used in sham operation group, but no coronary artery was ligated in rats to detect myocardial fine by Western blot. Cell TXNIP expression 1.5 was detected by small animal ultrasound and left ventricular catheterization for the determination of cardiac function in mice 1.6. Myocardial infarction area after ischemia and reperfusion was detected by Evan blue /TTC double staining method. The myocardial infarction area of mice was measured by TUNEL staining to determine the apoptosis of murine cardiomyocytes by TUNEL staining or whether TXNIP could affect the myocardial autophagy level 2.1 LC3-GFP mice were identified, and TXNIPMyosin6-Cre-LC3-GFP mice and LC3-GFP-TXNIP overexpressed mice 2.2 were used to prepare the model of myocardial ischemia and myocardial ischemia reperfusion in mice. The myocardial protein content was detected after 40 minutes of ischemia and 3 hours after reperfusion respectively. The electron microscopy and fluorescence microscopy were used after 40 minutes of reperfusion for 3 hours after ischemia. Detection of autophagy in mice 2.3 the expression of LC3 and P62 in myocardium was detected by Western blot 2.4 using transmission electron microscope to observe the autophagic corpuscle 2.5 using fluorescence microscope to observe the autophagic body LC3-GFP in mice 3 to explain the specific signaling pathway of TXNIP regulating autophagy 3.1 to identify AMPKfloxed/ floxedMyosin6-Cre mice and to construct AMPKfloxed/floxedMyo Sin6-Cre-TXNIP was overexpressed in mice 3.2 to prepare the model of myocardial ischemia and myocardial ischemia reperfusion model in mice. After 40 minutes of ischemia and 3 hours of reperfusion, the content of myocardial protein and the content of ATP were detected. The autophagy condition of mice was detected by electron microscope after 40 minutes of ischemia and 3 hours after ischemia, and the ATP content of myocardial tissue was measured by fluorescence method. Measurement of P AMPK, P AMPK, AMPK, P Raptor, Raptor, P ULK1, ULK1 expression 3.5 through the transmission electron microscope to observe the expression of myocardial autophagic body 3.6 through the transmission electron microscope (3.6) by Western blot TXNIP increased over expression of 1.2 TXNIP during the injury, which aggravated the decrease of cardiac function after myocardial ischemia and reperfusion in mice. TXNIP overexpressed mice significantly increased the left ventricular ejection fraction caused by myocardial ischemia reperfusion injury. After reperfusion, the three indexes of LVEDP, +dp /dtmax and -dp/dtmax in TXNIP overexpressed mice were significantly worse than WT Mice and TXNIP knockout mice. The above index of TXNIP knockout mice was further improved by 1.3 TXNIP over expression, which increased the infarct area and apoptosis after ischemia-reperfusion. Compared with the wild type mice, the TXNIP overexpressed mice significantly increased the dead area of myocardial infarction and the apoptosis of myocardial cells, while the TXNIP knockout was small. Ischemia reperfusion injury in rats was significantly less than 2 increased TXNIP increased the increase of 2.1 TXNIP of autophagy induced by myocardial ischemia reperfusion. The content of autophagy related protein in myocardial ischemia reperfusion after ischemia-reperfusion, the ratio of LC3 II/LC3 I in TXNIP overexpressed mice increased further, compared with wild type and TXNIP knockout mice. There were statistical differences. The ratio of LC3 II/LC3 I in wild type and TXNIP knockout mice was also statistically different, and the ratio of knockout mice was significantly lower by 2.2 TXNIP. The number of autophagic corpuscles in myocardial cells after ischemia-reperfusion was transmitted through transmission electron microscopy and observation of fluorescence spots in cardiac myocytes of LC3-GFP mice, and we found autophagic corpuscle. The level was significantly reduced in the TXNIP knockout mice, while the increase of 3 TXNIP in the TXNIP overexpressed mice was significantly increased by AMPK to regulate the increase of autophagy induced by myocardial ischemia and reperfusion by 3.1 TXNIP. The ATP generation in the myocardial ischemia reperfusion was compared with that of the wild type mice. The ATP content of the TXNIP knockout mice was significantly higher after the blood reperfusion, with a statistically significant difference. The ATP content of myocardium in TXNIP overexpressed mice was significantly decreased by 3.2 TXNIP through AMPK regulation of autophagy in TXNIP overexpressed mice. Ischemia reperfusion induced AMPK, Raptor phosphorylation activation increased, and TXNIP knockout mice could reduce AMPK, Raptor phosphorylation activation, and ULK1 phosphorylation inactivation. Further, AMPK was knocked out. Later, compared with the wild type mice with AMPK activity, the number of autophagic corpuscles resulting from TXNIP overexpression was significantly reduced. This topic confirmed that the increase of 1.TXNIP after ischemia-reperfusion caused the decrease of cardiac function, the increase of myocardial infarction area and the increase of apoptosis, while knockout TXNIP had the above mentioned above. In contrast,.2.TXNIP does not affect autophagy during myocardial ischemia, but increases autophagy at the stage of reperfusion, and its mechanism is mediated by AMPK. These results provide a new idea for our further understanding of the increase of autophagy induced by myocardial ischemia and reperfusion.

【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R54
，

本文編號：1895442