Prdx6对脑死亡状态下缺血缺氧性肝脏功能损伤保护作用的机制研究

发布时间：2018-06-03 02:28

  本文选题：过氧化物氧化还原酶6 + 脑死亡　； 参考：《武汉大学》2015年博士论文

【摘要】：第一部分背景：肝移植是治疗终末期肝功能衰竭的唯一有效方法。由于供体器官严重短缺,为扩大供体器官来源,越来越多的肝移植采用脑死亡供体肝脏。然而,与活体供肝肝移植相比,脑死亡供体肝移植术后器官功能差,术后并发症高,患者的生存率低。有多种原因会造成这种情况,其中,由于大量儿茶酚胺的释放,肝脏局部缺血缺氧,引起氧化应激而导致ROS增多是一个重要的因素。Prdx6蛋白是一个过氧化物酶家族蛋白,它能清除ROS及相关的过氧化物,保护机体免受内部或外部氧化应激的损伤。目的：研究脑死亡供体肝脏中肝细胞凋亡的发生情况及Prdx6蛋白的表达变化。方法：收集10例脑死亡器官捐献者的肝脏组织,收集6例肝血管瘤患者接受肝血管瘤切除手术切下的肝脏组织,通过TUNEL凋亡试剂盒检测确定脑死亡后肝脏凋亡情况,Western Blot法检测脑死亡供体肝脏中Prdx6蛋白及IκB、pIκB蛋白的表达量。结果：脑死亡供体肝脏中肝细胞的凋亡增加,Prdx6蛋白表达下降,NF-κB相关蛋白IκB表达下降、plκB表达上升。结论：脑死亡供体肝脏肝细胞凋亡增加可能与Prdx6蛋白表达下降有关,NF-κB可能参与Prdx6蛋白的表达。第二部分背景：Prdx6是一个双功能酶,除了具有过氧化物酶活性以外,它还具有PLA2酶活性；其具有抗氧化应激的功能。缺氧可刺激细胞线粒体产生ROS,造成细胞的损伤。在肝脏中,当肝细胞受到缺血再灌注引起的氧化应激刺激时,Prdx6可移位进入肝细胞的线粒体中发挥保护作用。本研究在脑死亡肝脏供体肝脏中发现Prdx6表达下降,肝细胞凋亡明显。现使用L02细胞进行氧糖剥夺处理建立缺血缺氧模型模拟脑死亡状态下肝脏的缺血缺氧状态进行进一步研究。目的：探讨Prdx6在缺血缺氧状态下对肝细胞的保护作用及其机制。方法：将L02细胞在缺血缺氧状态下分别培养6小时、12小时、18小时和24小时,收集细胞培养基检测其中的ALT、AST和LDH,使用ROS检测试剂盒检测细胞内ROS水平,CCK8试剂盒检测细胞活力,流式细胞仪检测L02细胞的凋亡,Western Blot检测细胞中Prdx6蛋白及IκB、p1κB蛋白的表达变化；构建pIRES2-ZsGreen1- Prdx6质粒转染L02细胞过表达Prdx6,再次检测Prdx6蛋白的表达、细胞的损伤情况及细胞内ROS水平。结果：缺血缺氧状态下细胞培养基内ALT、AST和LDH上升,细胞内ROS水平上升,细胞凋亡增加,细胞活力下降,Prdx6蛋白表达下降,NF-κB相关蛋白IκB表达下降、pIκB表达上升,且其变化程度随着缺血缺氧时间的延长而更明显。过表达Prdx6可以降低细胞内ROS水平,减轻细胞的缺血缺氧性损伤。结论：Prdx6蛋白在缺血缺氧状态下可通过降低细胞内ROS水平,减轻细胞损伤,发挥对细胞的保护作用。第三部分背景：NF-κB通过激活数以百计的目的基因对细胞的炎症反应、免疫应答以及细胞发育、生长、凋亡等多个过程起关键性作用。脑死亡状态下肝脏缺血缺氧,ROS生成增多,ROS可以通过非经典途径激活NF-KB,同时NF-KB与Prdx6蛋白的表达存在密切联系。本实验前两部分发现在脑死亡供体肝脏和缺血缺氧肝细胞中,NF-KB被激活。目的：探讨缺血缺氧状态下NF-κB对Prdx6蛋白的调控机制。方法：使用NF-κB特异性抑制剂BAY11-7082(5 μM)处理L02细胞1小时,并将细胞缺血缺氧状态下培养12小时,Western Blot检测细胞中Prdx6蛋白及IκB、pIκB蛋白的表达变化,收集细胞培养基检测其中的ALT、AST和LDH,CCK8试剂盒检测细胞活力。结果：BAY11-7082可以抑制NF-κB的激活,可使Prdx6蛋白表达上升,同时与对照组相比,细胞培养基内AST和LDH降低,细胞活力上升。结论：缺血缺氧状态下NF-KB激活,并负性调节Prdx6蛋白的表达。第四部分背景：Prdx6蛋白是一个独特的双功能酶,具有过氧化物酶活性和PLA2酶活性。ROS可以使细胞膜中的不饱和脂肪酸过氧化,损伤细胞膜,导致细胞受损。PLA2酶可以水解氧化的磷脂sn-2位置的酰基或烷基,参与损伤细胞膜的修复。目的：探讨缺血缺氧状态下PLA2酶活性对细胞的保护作用。方法：使用不同浓度(0μM,10μM,20μM,30μM,和50 μM)PLA2酶活性抑制剂MJ33处理L02细胞12小时或处理0.5小时后去除MJ33继续培养12小时,CCK8检测细胞的活力；MJ33预处理0.5小时后L02细胞缺血缺氧培养12小时(MJ33+0.5 h组),L02细胞接受MJ33与缺血缺氧同时处理12小时(MJ33+12 h组),CCK8检测两组细胞的活力,并收集20uM MJ33组的细胞培养基检测ALT、AST和LDH；PLA2酶活性的测定：细胞分三组(1)MJ33处理0.5小时后立即检测PLA2酶活性,(2)MJ33处理12小时后立即检测PLA2酶活性,(3)MJ33处理0.5小时后移除MJ33继续培养12小时,再检测PLA2酶活性。结果：不同浓度MJ33处理L02细胞12小时或处理0.5小时后去除MJ33继续培养12小时细胞活力无变化,表明MJ33在50uM浓度以下对L02细胞无毒性反应；不同浓度的MJ33处理肝细胞预处理0.5小时后使肝细胞处于缺血缺氧状态12小时,或MJ33与缺血缺氧同时处理12小时,两组细胞的活力均下降,且随着MJ33浓度升高,两组细胞活力下降程度加重,同时,MJ33+12 h组细胞的损伤程度比MJ33+0.5 h组细胞重；细胞培养基中AST和LDH明显上升,并且MJ33+12 h组细胞培养基中AST和LDH的上升幅度也更大；MJ33处理细胞0.5小时或12小时后立即检测PLA2酶活性,可见PLA2酶活性明显下降,MJ33处理肝细胞0.5小时后,去除MJ33再正常培养12小时,PLA2酶活性均有所恢复,且恢复程度与MJ33的浓度具有负相关性。结论：在缺血缺氧的环境下,Prdx6的PLA2酶活性具有保护细胞抵抗氧化应激的作用。
[Abstract]:Background: liver transplantation is the only effective method for the treatment of end-stage liver failure. Due to the severe shortage of donor organs, more and more liver transplantation uses brain death donor liver for the enlargement of donor organ sources. However, compared with living donor liver transplantation, the organ function of the donor liver transplantation is poor and the postoperative complications are high. The survival rate of the patients is low. There are many reasons for this, in which the release of a large number of catecholamines, partial ischemia and hypoxia in the liver, and oxidative stress, the increase of ROS is an important factor, the.Prdx6 protein is a peroxidase family protein, which can remove ROS and related peroxides to protect the body from inside the body. Objective: To study the occurrence of hepatocyte apoptosis in the liver of the brain dead donor and the changes in the expression of Prdx6 protein. Methods: to collect the liver tissues of 10 donors of brain death organs, collect 6 cases of hepatic hemangioma and receive liver hemangioma resection and cut off the liver tissue, by TUNEL apoptosis Kit. The apoptosis of liver after brain death was determined. The expression of Prdx6 protein, I kappa B and pI kappa B protein in the liver of the brain dead donor was detected by Western Blot method. Results: the apoptosis of liver cells in the liver of the brain dead donor increased, the expression of Prdx6 protein decreased, the expression of I kappa B in the NF- kappa B related protein decreased and the expression increased. Conclusion: brain death donor liver liver is fine. The increase of apoptosis may be related to the decrease of Prdx6 protein expression. NF- kappa B may participate in the expression of Prdx6 protein. Second background: Prdx6 is a bifunctional enzyme. Besides the activity of peroxidase, it also has the activity of PLA2 enzyme; it has the function of antioxidant stress. Hypoxia stimulates the mitochondrial production of cell mitochondria and causes the cell. In the liver, when the liver cells are stimulated by oxidative stress caused by ischemia-reperfusion, Prdx6 can transfer into the mitochondria of the liver cells to play a protective role. This study found that the expression of Prdx6 decreased in the liver donor liver of the brain and the apoptosis of the liver cells was obvious. The oxygen glucose deprivation treatment of L02 cells was used to establish the ischemic hypoxia model. Objective: To investigate the ischemic and anoxic state of liver in the state of simulated brain death. Objective: To explore the protective effect and mechanism of Prdx6 on the liver cells under the condition of ischemia and anoxia. Methods: L02 cells were cultured for 6 hours, 12 hours, 18 hours and 24, respectively, and collected cell culture medium to detect ALT, AST And LDH, ROS detection kit was used to detect the intracellular ROS level, CCK8 kit was used to detect cell viability, flow cytometry was used to detect the apoptosis of L02 cells. Western Blot was used to detect the expression of Prdx6 protein and I kappa B, P1 nuclear B protein. Results: the cell damage and the intracellular ROS level. Results: ALT, AST and LDH increased in the cell culture base of ischemia and hypoxia, the ROS level in the cells increased, the cell apoptosis increased, the cell vitality decreased, the expression of Prdx6 protein decreased, the expression of I kappa B in NF- kappa B related protein decreased, the pI kappa B expression rose, and the degree of change with the ischemic anoxia time Prolonged and more obvious. Overexpression of Prdx6 can reduce intracellular ROS level and reduce cell ischemic and anoxic damage. Conclusion: Prdx6 protein can reduce cell damage by lowering intracellular ROS level in ischemic and anoxic state, and play protective effect on cells. Third background: NF- kappa B is activated by hundreds of target genes by activating NF- kappa B The inflammatory response, immune response, and cell development, growth, apoptosis and other processes play a key role. The liver ischemia and hypoxia, the increase of ROS production, and the activation of NF-KB through non classical pathways in the brain death state, and the close relationship between the expression of NF-KB and the Prdx6 protein. The first two parts of the experiment found that the donor liver in the brain died. NF-KB was activated in the hypoxic and anoxic hepatocytes. Objective: To investigate the regulation mechanism of NF- kappa B on Prdx6 protein under ischemic and anoxic state. Methods: BAY11-7082 (5 u M), a specific inhibitor of NF- kappa B, was used to treat L02 cells for 1 hours, and the cells were cultured for 12 hours under the condition of hypoxia and hypoxia, and Western Blot was used to detect the proteins and kappa kappa protein. ALT, AST and LDH, CCK8 kits were collected to detect cell viability. Results: BAY11-7082 could inhibit the activation of NF- kappa B and increase the expression of Prdx6 protein. Compared with the control group, AST and LDH decreased in cell culture and cell viability increased. Conclusion: NF-KB activation in the ischemic and anoxic state. Negative regulation of the expression of Prdx6 protein. Fourth part background: Prdx6 protein is a unique bifunctional enzyme, with peroxidase activity and PLA2 enzyme activity.ROS can cause the peroxidation of unsaturated fatty acids in the cell membrane, damage the cell membrane, and cause the cell damage.PLA2 enzyme to hydrolyze the oxidized phospholipid sn-2 position acyl or alkyl, and participate in the hydrolysis of the oxidized phospholipid sn-2. Objective: To investigate the repair of the damaged cell membrane. Objective: To explore the protective effect of PLA2 enzyme activity under ischemic and anoxic state. Methods: using different concentrations (0 mu M, 10 mu M, 20 mu M, 30 mu M, and 50 micron M) PLA2 enzyme activity inhibitor MJ33 to treat L02 cells for 12 hours or 0.5 hours after treatment for 12 hours, CCK8 detection of cell viability; MJ33 precondition After 0.5 hours, L02 cells were cultured for 12 hours of ischemia and hypoxia (group MJ33+0.5 h), L02 cells were treated with MJ33 and ischemic anoxia for 12 hours (MJ33+12 H Group). CCK8 detected the activity of two groups of cells, and collected the cell culture basis of 20uM MJ33 group, ALT, AST and viability: three groups of cells (1) were treated for 0.5 hours after 0.5 hours. The activity of PLA2 enzyme was detected (2) after 12 hours treatment, the activity of PLA2 enzyme was detected immediately after 12 hours. (3) after 0.5 hours of MJ33 treatment, MJ33 was removed for 12 hours, and then the activity of PLA2 enzyme was detected. The results showed that MJ33 treated L02 cells for 12 hours or after 0.5 hours to remove MJ33 to continue culture for 12 hours without change, indicating that MJ33 was below 50uM concentration. Nontoxic reaction to L02 cells; after 0.5 hours of pretreatment with different concentrations of MJ33, the liver cells were in the state of ischemia and hypoxia for 12 hours, or MJ33 was treated with ischemia and hypoxia for 12 hours, and the vitality of the two groups decreased, and as the concentration of MJ33 increased, the decrease of cell viability in the two groups was aggravated, and the loss of cells in the MJ33+12 H group was at the same time. The degree of injury was heavier than that of the MJ33+0.5 H group; AST and LDH increased obviously in the cell culture medium, and the increase of AST and LDH in the cell culture medium of MJ33+12 H group was also greater; MJ33 treated cells immediately detected the activity of PLA2 enzyme after 0.5 hours or 12 hours, which showed that the activity of PLA2 enzyme decreased obviously. After 0.5 hours' MJ33 treated hepatocytes, it removed normal and then normal. The activity of PLA2 enzyme was recovered for 12 hours, and the degree of recovery was negatively correlated with the concentration of MJ33. Conclusion: in the environment of ischemic and anoxia, the activity of PLA2 enzyme of Prdx6 can protect the cells against oxidative stress.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R657.3

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