本文選題：過氧化物氧化還原酶6 + 腦死亡��； 參考：《武漢大學(xué)》2015年博士論文

【摘要】：第一部分背景：肝移植是治療終末期肝功能衰竭的唯一有效方法。由于供體器官嚴(yán)重短缺,為擴大供體器官來源,越來越多的肝移植采用腦死亡供體肝臟。然而,與活體供肝肝移植相比,腦死亡供體肝移植術(shù)后器官功能差,術(shù)后并發(fā)癥高,患者的生存率低。有多種原因會造成這種情況,其中,由于大量兒茶酚胺的釋放,肝臟局部缺血缺氧,引起氧化應(yīng)激而導(dǎo)致ROS增多是一個重要的因素。Prdx6蛋白是一個過氧化物酶家族蛋白,它能清除ROS及相關(guān)的過氧化物,保護機體免受內(nèi)部或外部氧化應(yīng)激的損傷。目的：研究腦死亡供體肝臟中肝細(xì)胞凋亡的發(fā)生情況及Prdx6蛋白的表達(dá)變化。方法：收集10例腦死亡器官捐獻者的肝臟組織,收集6例肝血管瘤患者接受肝血管瘤切除手術(shù)切下的肝臟組織,通過TUNEL凋亡試劑盒檢測確定腦死亡后肝臟凋亡情況,Western Blot法檢測腦死亡供體肝臟中Prdx6蛋白及IκB、pIκB蛋白的表達(dá)量。結(jié)果：腦死亡供體肝臟中肝細(xì)胞的凋亡增加,Prdx6蛋白表達(dá)下降,NF-κB相關(guān)蛋白IκB表達(dá)下降、plκB表達(dá)上升。結(jié)論：腦死亡供體肝臟肝細(xì)胞凋亡增加可能與Prdx6蛋白表達(dá)下降有關(guān),NF-κB可能參與Prdx6蛋白的表達(dá)。第二部分背景：Prdx6是一個雙功能酶,除了具有過氧化物酶活性以外,它還具有PLA2酶活性；其具有抗氧化應(yīng)激的功能。缺氧可刺激細(xì)胞線粒體產(chǎn)生ROS,造成細(xì)胞的損傷。在肝臟中,當(dāng)肝細(xì)胞受到缺血再灌注引起的氧化應(yīng)激刺激時,Prdx6可移位進入肝細(xì)胞的線粒體中發(fā)揮保護作用。本研究在腦死亡肝臟供體肝臟中發(fā)現(xiàn)Prdx6表達(dá)下降,肝細(xì)胞凋亡明顯�，F(xiàn)使用L02細(xì)胞進行氧糖剝奪處理建立缺血缺氧模型模擬腦死亡狀態(tài)下肝臟的缺血缺氧狀態(tài)進行進一步研究。目的：探討Prdx6在缺血缺氧狀態(tài)下對肝細(xì)胞的保護作用及其機制。方法：將L02細(xì)胞在缺血缺氧狀態(tài)下分別培養(yǎng)6小時、12小時、18小時和24小時,收集細(xì)胞培養(yǎng)基檢測其中的ALT、AST和LDH,使用ROS檢測試劑盒檢測細(xì)胞內(nèi)ROS水平,CCK8試劑盒檢測細(xì)胞活力,流式細(xì)胞儀檢測L02細(xì)胞的凋亡,Western Blot檢測細(xì)胞中Prdx6蛋白及IκB、p1κB蛋白的表達(dá)變化；構(gòu)建pIRES2-ZsGreen1- Prdx6質(zhì)粒轉(zhuǎn)染L02細(xì)胞過表達(dá)Prdx6,再次檢測Prdx6蛋白的表達(dá)、細(xì)胞的損傷情況及細(xì)胞內(nèi)ROS水平。結(jié)果：缺血缺氧狀態(tài)下細(xì)胞培養(yǎng)基內(nèi)ALT、AST和LDH上升,細(xì)胞內(nèi)ROS水平上升,細(xì)胞凋亡增加,細(xì)胞活力下降,Prdx6蛋白表達(dá)下降,NF-κB相關(guān)蛋白IκB表達(dá)下降、pIκB表達(dá)上升,且其變化程度隨著缺血缺氧時間的延長而更明顯。過表達(dá)Prdx6可以降低細(xì)胞內(nèi)ROS水平,減輕細(xì)胞的缺血缺氧性損傷。結(jié)論：Prdx6蛋白在缺血缺氧狀態(tài)下可通過降低細(xì)胞內(nèi)ROS水平,減輕細(xì)胞損傷,發(fā)揮對細(xì)胞的保護作用。第三部分背景：NF-κB通過激活數(shù)以百計的目的基因?qū)?xì)胞的炎癥反應(yīng)、免疫應(yīng)答以及細(xì)胞發(fā)育、生長、凋亡等多個過程起關(guān)鍵性作用。腦死亡狀態(tài)下肝臟缺血缺氧,ROS生成增多,ROS可以通過非經(jīng)典途徑激活NF-KB,同時NF-KB與Prdx6蛋白的表達(dá)存在密切聯(lián)系。本實驗前兩部分發(fā)現(xiàn)在腦死亡供體肝臟和缺血缺氧肝細(xì)胞中,NF-KB被激活。目的：探討缺血缺氧狀態(tài)下NF-κB對Prdx6蛋白的調(diào)控機制。方法：使用NF-κB特異性抑制劑BAY11-7082(5 μM)處理L02細(xì)胞1小時,并將細(xì)胞缺血缺氧狀態(tài)下培養(yǎng)12小時,Western Blot檢測細(xì)胞中Prdx6蛋白及IκB、pIκB蛋白的表達(dá)變化,收集細(xì)胞培養(yǎng)基檢測其中的ALT、AST和LDH,CCK8試劑盒檢測細(xì)胞活力。結(jié)果：BAY11-7082可以抑制NF-κB的激活,可使Prdx6蛋白表達(dá)上升,同時與對照組相比,細(xì)胞培養(yǎng)基內(nèi)AST和LDH降低,細(xì)胞活力上升。結(jié)論：缺血缺氧狀態(tài)下NF-KB激活,并負(fù)性調(diào)節(jié)Prdx6蛋白的表達(dá)。第四部分背景：Prdx6蛋白是一個獨特的雙功能酶,具有過氧化物酶活性和PLA2酶活性。ROS可以使細(xì)胞膜中的不飽和脂肪酸過氧化,損傷細(xì)胞膜,導(dǎo)致細(xì)胞受損。PLA2酶可以水解氧化的磷脂sn-2位置的�；蛲榛�,參與損傷細(xì)胞膜的修復(fù)。目的：探討缺血缺氧狀態(tài)下PLA2酶活性對細(xì)胞的保護作用。方法：使用不同濃度(0μM,10μM,20μM,30μM,和50 μM)PLA2酶活性抑制劑MJ33處理L02細(xì)胞12小時或處理0.5小時后去除MJ33繼續(xù)培養(yǎng)12小時,CCK8檢測細(xì)胞的活力；MJ33預(yù)處理0.5小時后L02細(xì)胞缺血缺氧培養(yǎng)12小時(MJ33+0.5 h組),L02細(xì)胞接受MJ33與缺血缺氧同時處理12小時(MJ33+12 h組),CCK8檢測兩組細(xì)胞的活力,并收集20uM MJ33組的細(xì)胞培養(yǎng)基檢測ALT、AST和LDH；PLA2酶活性的測定：細(xì)胞分三組(1)MJ33處理0.5小時后立即檢測PLA2酶活性,(2)MJ33處理12小時后立即檢測PLA2酶活性,(3)MJ33處理0.5小時后移除MJ33繼續(xù)培養(yǎng)12小時,再檢測PLA2酶活性。結(jié)果：不同濃度MJ33處理L02細(xì)胞12小時或處理0.5小時后去除MJ33繼續(xù)培養(yǎng)12小時細(xì)胞活力無變化,表明MJ33在50uM濃度以下對L02細(xì)胞無毒性反應(yīng)；不同濃度的MJ33處理肝細(xì)胞預(yù)處理0.5小時后使肝細(xì)胞處于缺血缺氧狀態(tài)12小時,或MJ33與缺血缺氧同時處理12小時,兩組細(xì)胞的活力均下降,且隨著MJ33濃度升高,兩組細(xì)胞活力下降程度加重,同時,MJ33+12 h組細(xì)胞的損傷程度比MJ33+0.5 h組細(xì)胞重；細(xì)胞培養(yǎng)基中AST和LDH明顯上升,并且MJ33+12 h組細(xì)胞培養(yǎng)基中AST和LDH的上升幅度也更大；MJ33處理細(xì)胞0.5小時或12小時后立即檢測PLA2酶活性,可見PLA2酶活性明顯下降,MJ33處理肝細(xì)胞0.5小時后,去除MJ33再正常培養(yǎng)12小時,PLA2酶活性均有所恢復(fù),且恢復(fù)程度與MJ33的濃度具有負(fù)相關(guān)性。結(jié)論：在缺血缺氧的環(huán)境下,Prdx6的PLA2酶活性具有保護細(xì)胞抵抗氧化應(yīng)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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R657.3

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