TXNIP在心肌缺血再灌注中的作用研究

发布时间：2018-05-16 04:24

  本文选题：TXNIP + 心肌缺血/再灌注　； 参考：《第四军医大学》2015年博士论文

【摘要】：研究背景心肌缺血后恢复血流可显著减轻心肌损伤。然而血管的开通却会发生缺血再灌注损伤1,2。进一步的研究表明,心肌细胞的自噬在缺血再灌注损伤中起到重要作用。正常情况下,心脏维持着低水平的自噬。心肌缺血时自噬升高,具有对抗缺血的保护作用。然而在再灌注期,本应下降的自噬却继续升高,损害了心脏功能并促进了心肌细胞损伤和死亡3。缺血再灌注时血流既已恢复,可自噬因何会继续激活升高并损害心肌,其机制我们仍不清楚。TXNIP,又称Trx相互结合蛋白4-6,在心肌缺血时表达升高。TXNIP敲除小鼠心肌缺血再灌注时,由于TXNIP的敲除导致细胞的能量代谢更倾向于无氧糖酵解,避免缺血所导致的线粒体进行的无效的氧化磷酸化,生成了更多的ATP7。既往报道,当细胞缺乏能量时,AMPK激活导致自噬8。既然TXNIP能够调控心肌细胞再灌注时期的能量代谢,且又在此时表达过度升高,TXNIP是否是心肌缺血再灌注时调控自噬的分子呢,我们仍不清楚。因此,在后续实验中我们围绕此进行了系列实验,以期证明缺血再灌注时TXNIP增加参与调控了自噬水平的提高。研究目的1.观察过表达或敲除TXNIP是否影响MI/R后心肌损伤2.研究过表达或敲除TXNIP是否会影响心肌自噬水平3.阐述TXNIP调控自噬的具体信号通路研究方法1观察过表达或敲除TXNIP是否影响MI/R后心肌损伤1.1构建并鉴定TXNIPfloxed/floxed Myosin6-Cre小鼠1.2通过心肌点注射腺病毒构建过表达TXNIP的小鼠1.3制备小鼠心肌缺血及心肌缺血再灌注模型用6-0丝线结扎小鼠左冠状动脉后,将丝线打活结造成心肌缺血,缺血40分钟后打开活结。分别在缺血40分钟后、再灌注1.5小时及再灌注3小时后检测心肌蛋白含量。在缺血40分钟再灌注3小时后检测心肌细胞凋亡,在再灌注24小时后检测心功能及心肌梗死面积。假手术组采用同样的手术方法,但并不结扎小鼠冠状动脉1.4通过Western blot检测心肌细胞TXNIP表达1.5通过小动物超声检测及左心室内插管测定小鼠小鼠心脏功能1.6通过伊文氏蓝/TTC双染法检测小鼠心肌缺血再灌注后心肌梗死面积1.7通过TUNEL染色测定小鼠心肌细胞凋亡2研究过表达或敲除TXNIP是否会影响心肌自噬水平2.1鉴定LC3-GFP小鼠,同时构建TXNIPMyosin6-Cre-LC3-GFP小鼠及LC3-GFP-TXNIP过表达小鼠2.2制备小鼠心肌缺血及心肌缺血再灌注模型方法同前。分别在缺血40分钟后及再灌注3小时后检测心肌蛋白含量。在缺血40分钟再灌注3小时后使用电镜及荧光显微镜检测小鼠自噬情况2.3通过Western blot检测心肌组织LC3及P62的表达2.4使用透射电镜观察心肌自噬小体2.5使用荧光显微镜观察小鼠LC3-GFP自噬小体3阐述TXNIP调控自噬的具体信号通路3.1鉴定AMPKfloxed/floxedMyosin6-Cre小鼠,构建AMPKfloxed/floxedMyosin6-Cre-TXNIP过表达小鼠3.2制备小鼠心肌缺血及心肌缺血再灌注模型方法同前。分别在缺血40分钟后及再灌注3小时后检测心肌蛋白含量及ATP含量。在缺血40分钟再灌注3小时后使用电镜检测小鼠自噬情况3.3通过荧光法测定心肌组织ATP含量3.4通过Western blot检测心肌细胞p AMPK、AMPK、p Raptor、Raptor、p ULK1、ULK1的表达3.5通过透射电镜观察相关小鼠心肌自噬小体3.6通过Western blot检测心肌组织LC3表达研究结果1缺血所致的TXNIP升高加重心肌缺血再灌注损伤1.1心肌缺血及再灌注损伤过程中TXNIP持续升高1.2 TXNIP过表达加重了小鼠心肌缺血再灌注后的心脏功能降低TXNIP过表达小鼠显著加重了心肌缺血再灌注损伤导致的左室射血分数降低。缺血再灌后,TXNIP过表达小鼠的LVEDP,+dp/dtmax和-dp/dtmax三个指标均明显差于WT小鼠与TXNIP敲除小鼠。而TXNIP敲除小鼠的上述指标相比野生小鼠出现进一步的好转1.3 TXNIP过表达增加了缺血再灌注后的心肌梗死面积及细胞凋亡与野生型小鼠相比,TXNIP过表达小鼠显著增加心肌梗死面积及心肌细胞凋亡,而TXNIP敲除小鼠缺血再灌注损伤则显著更轻2升高的TXNIP加强了心肌缺血再灌所导致的自噬增加2.1 TXNIP影响了缺血再灌注后心肌自噬相关蛋白的含量在缺血再灌注后,TXNIP过表达小鼠的LC3 II/LC3 I的比值进一步升高,与野生型小鼠和TXNIP敲除鼠相比均出现统计学差异。野生型小鼠与TXNIP敲除鼠的LC3 II/LC3 I的比值亦出现统计学差异,敲除鼠的比值显著更低2.2 TXNIP影响了缺血再灌注后心肌细胞的自噬小体数量通过透射电镜及观察LC3-GFP小鼠的心肌细胞荧光斑点,我们发现自噬小体水平在TXNIP敲除鼠中显著降低,而在TXNIP过表达小鼠中显著升高3 TXNIP通过AMPK调控心肌缺血再灌导致的自噬增加3.1 TXNIP影响心肌缺血再灌注后ATP生成与野生型小鼠相比较,TXNIP敲除鼠的ATP含量在缺血再灌注后显著更高,具有统计学差异,而TXNIP过表达小鼠的心肌ATP含量出现了显著的下降3.2 TXNIP通过AMPK调节自噬在TXNIP过表达小鼠中,缺血再灌注导致AMPK、Raptor的磷酸化激活增加,而TXNIP敲除小鼠则可减少AMPK、Raptor的磷酸化激活,增加ULK1的磷酸化失活。进一步,在AMPK被敲除后,与具有AMPK活性的野生型小鼠相比,TXNIP过表达所导致的自噬小体数量显著减少研究结论本课题证实了:1.TXNIP在缺血再灌注后的升高,且这种升高导致心脏功能的降低,心肌梗死面积的增加及细胞凋亡的增多。而敲除TXNIP则具有上述相反得作用。2.TXNIP并不影响心肌缺血期的自噬,而却在再灌注期的升高增加了自噬,且其机制是通过AMPK所介导的。这些结果为我们进一步认识心肌缺血再灌注导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.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R54
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本文编号：1895442