小鼠肝缺血/再灌注损伤时肝脏Kupffer细胞中TLR2的表达

目的　观察小鼠肝部分缺血 /再灌注损伤时肝脏Kupffer细胞表面TLR2mRNA的表达及蛋白质的合成情况。方法　制作小鼠肝部分缺血 /再灌注损伤模型。采用原位灌注消化法分离并纯化Kupffer细胞 ,用大鼠抗小鼠TLR 2IgG和异硫氰酸荧光素 (FITC )的二抗进行染色 ,流式细胞仪 (FCM )测定阳性细胞数 ;并测定Kupffer细胞的TLR2mRNA含量。结果　缺血 60min ,再灌注 4h时 ,实验组小鼠肝脏Kupffer细胞表面TLR2mRNA表达及蛋白的合成量均明显高于假手术组 ,且缺血叶高于非缺血叶。结论　小鼠肝缺血 /再灌注损伤时 ,肝脏Kupffer细胞表面的TLR2mRNA表达明显增高 ,其蛋白质的水平也明显升高     

异丙酚对缺氧复氧诱导大鼠肝脏Kupffer细胞释放TNF-α、IL-1β和NO的影响

异丙酚是麻醉诱导与维持常用的静脉麻醉药物之一.研究表明异丙酚可减轻肝脏缺血再灌注损伤.肝脏缺血再灌注时可诱发单核巨噬细胞释放肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和一氧化氮(NO)等细胞因子,从而导致组织损伤.肝脏Kupffer细胞是位于肝窦内的巨噬细胞.本研究拟通过观察异丙酚对缺氧复氧诱导大鼠肝脏Kupffer细胞释放TNF-α、IL-1β和NO的影响,探讨其减轻肝脏缺血再灌注损伤的机制.     

大鼠肝内胆管上皮缺血再灌注损伤及丹参的保护作用

在多种肝胆手术特别是肝移植中不可避免地会出现肝脏的缺血再灌注损伤问题，国内外同行对肝脏在冷缺血或热缺血及再灌注后肝细胞、肝窦内皮细胞和Kupffer细胞等病理生理变化和损伤防护方面均做了大量研究工作，但对胆道系统的缺血再灌注损伤却极少有报道。然而，肝内胆管的缺血再灌注损伤及其导致的术后并发症，如胆管炎、肝内胆汁淤积、胆管狭窄、胆管消失综合征等，严重影响着术后肝脏的功能和移植物的存活。     

活性氧在肝脏缺血再灌注损伤中的研究进展

目的总结活性氧在肝脏缺血再灌注损伤中的发病机理及活性氧防治的最新研究进展。方法通过对CNKI、Pub Med等数据库文献进行检索,就活性氧在肝脏缺血再灌注损伤中的产生、损伤机理以及对近年来在活性氧防治方面的研究进展进行综述。结果在肝脏缺血再灌注损伤中,多形核白细胞、Kupffer细胞、线粒体以及肝组织中的酶产生大量的活性氧。活性氧主要通过破坏细胞膜上寡糖链中的糖分子、体内的不饱和脂肪酸、蛋白质分子、遗传物质、线粒体等导致细胞损伤甚至死亡。目前主要的防治方法为利用酶、维生素、中草药等清除活性氧,减轻肝脏缺血再灌注损伤。结论目前关于活性氧在肝脏缺血再灌注损伤中的研究取得了重要进展,针对活性氧对肝脏造成的损伤也提出了多种可行的防治方法,但要应用于临床还有待进一步深入研究。     

caspase蛋白在肝脏缺血再灌注损伤中的研究进展

caspase蛋白在促进细胞凋亡过程中发挥关键作用,在肝脏缺血再灌注损伤和对细胞凋亡过程的实验研究时,常可检测到caspase蛋白.现就caspase蛋白在肝脏缺血再灌注损伤中的研究进展作一综述.     

RNA干扰Kupffer细胞肿瘤坏死因子-α减轻大鼠肝脏缺血再灌注损伤

目的 观察RNA干扰肝脏Kupffer细胞肿瘤坏死因子-α(TNF-α)对大鼠肝脏缺血再灌注损伤的保护作用.方法 构建针对大鼠TNF-α基因的短发夹状RNA(shRNA)真核表达载体.肝脏缺血再灌注损伤前48 h经门静脉注射磷酸盐缓冲液(PBS)、空载体或TNF-α shRNA.实验随机分为4组,假手术组、PBS组、空载体组和shRNA组.阻断大鼠70%入肝血流40 min,再灌注6 h检测血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、肝脏Kupffer细胞TNF-α mRNA、血清TNF-α、肝组织中丙二醛(MDA)以及超氧化物岐化酶(SOD)含量.结果 与PBS组和空载体组比较,shRNA组再灌注6 h后血清ALT和AST水平显著降低(P＜0.05),Kupffer细胞TNF-α mRNA水平、血清TNF-α水平(56.6±6.7 pg/ml比87.8±8.7 pg/ml和96.5±7.3 pg/ml,P＜0.05)、肝组织中MDA含量(93.4±13.3 nmol/mg比133.5±12.4 nmo1/mg和136.7±13.6 nmol/mg,P＜0.05)显著降低,SOD活性显著升高(22.4±4.6 U/mg比12.2±3.1 U/mg和11.4±2.9 U/mg,P＜0.05).结论 RNA干扰Kupffer细胞TNF-α基因的表达可以减轻大鼠肝脏缺血再灌注损伤.     

枯否细胞和肝脏缺血再灌注损伤

目的 了解枯否细胞在肝脏缺血再灌注损伤中的作用。方法 采用文献回顾的方法对枯否细胞在肝脏缺血再灌注损伤中的作用加以综述。结果 活化后的枯否细胞可产生和释放多种介质直接或间接地影响肝脏微循环。结论 枯否细胞在肝缺血再灌注损伤中发挥了重要作用。     

大鼠肝移植缺血再灌注后Kupffer细胞CD14基因及蛋白的表达

目的研究大鼠肝移植缺血再灌注后Kupffer细胞CD14基因及蛋白的表达,探讨其在再灌注损伤中的作用.方法分离培养大鼠肝移植缺血再灌注后0（对照组）、2、6、12 h（IR组）的Kupffer细胞,用逆转录聚合酶联反应（RT-PCR）检测Kupffer细胞CD14 mRNA的表达,用免疫印迹检测CD14蛋白合成,用酶联免疫吸附试验（ELISA）法测定培养上清TNF-α的分泌量.然后在上述时间点的细胞培养液中加入抗CD14抗体（anti-CD14组）,观察CD14抗体对TNF-α分泌的影响.结果再灌注后Kupffer细胞CD14 mRNA、蛋白以及TNF-α随观察时间点呈逐步上升趋势（与对照组相比,P＜0.01）.应用抗CD14抗体后,TNF-α表达较IR组明显降低（P＜0.01）.结论再灌注后Kupffer细胞CD14基因及蛋白的表达明显升高,TNF-α的合成和分泌也明显增强;抗CD14单抗能明显抑制TNF-α的产生;CD14在介导Kupffer细胞激活和肝移植缺血再灌注损伤中可能起重要作用.     

肝脏缺血后处理的保护作用机制

肝脏缺血后处理是指肝脏在长时间缺血后,在再灌注之前进行一次或数次短暂重复的缺血再灌注,能提高肝脏对长时间缺血的耐受性,减轻缺血再灌注损伤.近几年被证实为一种有效、可控制的新的减轻再灌注损伤的方法.肝脏缺血后处理的保护机制与保护肝窦内皮和肝脏细胞超微结构,减轻活性氧引起的细胞损伤及炎症反应,减轻细胞内及线粒体内钙超载,调控凋亡基因,改变线粒体离子通道开放状态等有关.本文就缺血后处理的机制作一简要综述.     

丙泊酚与肝脏缺血/再灌注损伤

缺血/再灌注对肝脏造成损伤.众多资料显示丙泊酚对肝脏缺血/再灌注损伤有保护作用,这一保护作用与其抗氧化,阻断钙超载,减轻炎性细胞导致的损伤有关.肝脏缺血/再灌注也影响了丙泊酚的代谢.     

Ischemia and reperfusion injury in liver transplantation

Ischemia/reperfusion (I/R) injury is a multifactorial process detrimental to liver graft function. An understanding of the mechanisms involved in I/R injury is essential for the design of therapeutic strategies to improve the outcome of liver transplantation. The generation of reactive oxygen species subsequent to reoxygenation inflicts tissue damage and initiates a cellular cascade leading to inflammation, cell death, and ultimate organ failure. The accruing evidence suggests that Kupffer cells and T cells mediate the activation of neutrophil inflammatory responses. Activated neutrophils infiltrate the injured liver in parallel with increased expression of adhesion molecules on endothelial cells. The heme oxygenase (HO) system is among the most critical of the cytoprotective mechanisms activated during the cellular stress, exerting anti-oxidant and anti-inflammatory functions, modulating the cell cycle, and maintaining the microcirculation. The activation of toll-like receptors (TLR) on Kupffer cells may provide the triggering signal for pro-inflammatory responses in the I/R injury sequence. Indeed, dissecting TLR downstream signaling pathways plays a fundamental role in exploring novel therapeutic strategies based on the concept that hepatic I/R injury represents a case for host "innate" immunity.     

枯否细胞在肝移植手术后缺血再灌注损伤中的作用

目的总结枯否细胞在肝移植术后缺血再灌注损伤中的作用。方法通过复习文献的方法对枯否细胞在缺血再灌注损伤中的作用进行综述。结果枯否细胞是肝内固有的巨噬细胞,肝移植手术后枯否细胞被激活释放一系列炎症介质,包括细胞因子、活性氧中间产物、趋化因子等,启动缺血再灌注损伤(ischemia reperfusioninjury,IRI),使移植肝失功。同时,枯否细胞不仅可以通过释放NO来减轻缺血再灌注损伤,也可以特异性的产生血红素加氧酶-1(heme oxygenase-1,HO-1)来发挥对缺血再灌注损伤的保护作用,并且有研究表明HO-1降解血红素产生的代谢产物一氧化碳(carbon monoxide,CO)也有同样的作用。结论枯否细胞在肝移植术后可以发挥双向性作用,如何减少枯否细胞释放各种有害物质,增加有利物质的表达,是今后预防移植肝后缺血再灌注损伤研究的关键。     

血红素氧合酶-1减轻大鼠移植肝缺血再灌注损伤的作用及机制

目的 探讨血红素氧合酶-1(HO-1)减轻大鼠移植肝缺血再灌注损伤的作用及其机制.方法 选择近交系雄性SD大鼠作为肝移植的供、受者;采用单纯随机方法将48只SD大鼠随机分为对照组、抑制组和诱导组(每组供、受者各8只).对照组:供肝不用任何药物处理;抑制组:在获取供肝前24 h,经供者腹腔注射HO-1抑制剂锌原卟啉20 mg/kg进行预处理;诱导组:在获取供肝前24 h,经供者腹腔注射HO-1诱导剂钴原卟啉5 mg/kg进行预处理.获取供肝后,在4℃UW液中冷保存24 h.肝移植前检测供肝HO-1的表达水平;肝移植后6 h采血并获取移植肝标本,分离培养枯否细胞;检测受者的肝功能;检测枯否细胞培养上清中肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)的含量;观察移植肝组织病理学表现以及枯否细胞CD14 mRNA的表达水平和蛋白含量测定.结果 移植前诱导组供肝HO-1的表达水平明显增高.移植后诱导组血清丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)含量明显降低;移植肝组织病理学损伤减轻;枯否细胞培养上清中TNF-α和IL-6含量减少;而且枯否细胞上的CD14 mRNA表达水平和蛋白含量也明显低于抑制组.结论 诱导供肝HO-1表达上调可能抑制了枯否细胞的激活,从而减轻大鼠移植肝缺血再灌注损伤.     

HEGPOL: Randomized,placebo controlled,multicenter, double-blind clinical trial to investigate hepatoprotective effects of glycine in the postoperative phase of liver transplantation [ISRCTN69350312]

Background  

Kupffer cell-dependent ischemia / reperfusion (I/R) injury after liver transplantation is still of high clinical relevance, as it is strongly associated with primary dysfunction and primary nonfunction of the graft. Glycine, a non-toxic, non-essential amino acid has been conclusively shown in various experiments to prevent both activation of Kupffer cells and reperfusion injury. Based on both experimental and preliminary clinical data this study protocol was designed to further evaluate the early effect of glycine after liver transplantation.     

Taurine improves graft survival after experimental liver transplantation.

Relevant mechanisms of reperfusion injury after liver transplantation are most likely mediated by activated Kupffer cells. Recently, it has been demonstrated that taurine prevents Kupffer cell-activation in vitro. Thus, this study was designed to assess the effects of taurine after liver transplantation. Female Sprague-Dawley rats (210-240 g) were infused with taurine dissolved in normal saline, before organ harvest. Controls were infused with the same volume of normal saline without taurine. Following 4 hours of cold ischemia, liver transplantation was performed. Graft and animal survival, serum transaminases, liver histology, perfusion data of intravital microscopy, blood distribution at reperfusion, and both phagocytosis of Kupffer cells and expression of tumor necrosis factor alpha (TNF-alpha) to index cellular activation were investigated. For comparison, both, analysis of variance (ANOVA) and Fisher's exact test were used as appropriate. Results are presented as mean +/- SEM. Controls survived in 60% of cases. Taurine improved survival in a dose-dependent manner to 100% (P < 0.05). In controls, mean aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactic dehydrogenase (LDH) serum levels increased to 3,260 +/- 814; 1,703 +/- 432; and 14,071 +/- 3,177 U/L, respectively, after transplantation. In contrast, these values were between 20 and 45% of control values after taurine (P < 0.05). Histology taken after transplantation confirmed the significant protective effects of taurine, including the reduction of TNF-alpha expression. Time until homogeneous reperfusion of the graft improved to 50% of control values (P < 0.05). Further, taurine significantly decreased both phagocytosis of latex beads by Kupffer cells and leukocyte-endothelial cell interaction. In parallel, flow velocity of red blood cells as well as acinar and sinusoidal perfusion improved (P < 0.05). In conclusion, these data show for the first time in vivo that taurine minimizes reperfusion injury after liver transplantation. Decreased leukocyte-endothelial cell interaction and improved microcirculation are the proposed mechanisms, which are most likely Kupffer cell-dependent.     

Protection by pentoxifylline against graft failure from storage injury after orthotopic rat liver transplantation with arterialization

Destruction of the endothelial cell lining and activation of Kupffer cells after reperfusion limits the safe storage of livers for transplantation surgery. Tumor necrosis factor-alpha (TNF) release by activated Kupffer cells may contribute to graft failure from storage injury. Accordingly, we evaluated whether pentoxifylline, which suppresses macrophage TNF release, would improve graft survival after orthotopic rat liver transplantation with arterialization. Livers from syngeneic Lewis rats were stored for 12–24 h in cold UW solution. Prior to implantation, the livers were flushed with cold Ringer's solution or warm Carolina rinse solution B. With either rinse, pentoxifylline treatment of graft recipients significantly improved graft survival. Combined use of pentoxifylline (50 mg/kg for 5 days) and Carolina rinse solution doubled the safe storage time to 24 h. Acidotic pH and antioxidants were essential components of Carolina rinse solution that acted synergistically with pentoxifylline. Pentoxifylline was also shown to suppress TNF release by lipopolysaccharide (LPS)-stimulated cultured rat Kupffer cells. Thus, pentoxifylline may protect against primary non-function and failure of grafts from storage injury by suppressing excessive TNF release by activated Kupffer cells. However, neutralization of TNF with excess anti-TNF antibody did not improve survival. This may mean that depletion of TNF is as deleterious as excess TNF production. Alternatively, other Kupffer cell secretions [e.g., interleukin-1 (IL-1), interleukin-6 (IL-6) and other cytokines] may be involved in the pathogenesis of graft failure. In conclusion, pentoxifylline could protect against graft failure from storage injury.     

Elevation of serum interleukin-18 levels and activation of Kupffer cells in biliary atresia

BACKGROUND/PURPOSE: Interleukin-18 (IL-18)/interferon-gamma-inducing factor (IGIF) is a novel proinflammatory cytokine that can induce interferon gamma (IFN-gamma). In addition, IL-18 enhances intracellular adhesion molecule-1 (ICAM-1) expression as well as Fas ligand (FasL) expression, and induces apoptosis in hepatic injury. The aim of this study was to clarify the potential role of IL-18 in the pathogenesis of the progressive inflammation and fibrosis in biliary atresia (BA). METHODS: Six children with BA before hepatic portoenterostomy (HPE), 13 with BA including 7 without jaundice and 6 with persistent jaundice after HPE, and 16 healthy controls were examined. Blood samples were obtained preoperatively from 6 patients, after HPE from 13, and after liver transplantation from 4. The IL-18 level was determined by an enzyme-linked immunosorbent assay (ELISA). Immunohistochemically, liver specimens from BA patients were studied using a monoclonal antibody to macrophage-associated antigen (CD68). RESULTS: IL-18 levels were elevated in the patients before HPE compared with those of the controls (349+/-54 pg/mL v. 138+/-13 pg/mL, P<.0001). After HPE, extremely high concentrations of IL-18 were observed in patients with persistent jaundice (532+/-95 pg/mL, P<.0001), and the IL-18 levels were significantly high even in the patients without jaundice (249+/-29 pg/mL, P<0.005). The high IL-18 level lasted for a long time even in the patients without jaundice after HPE. In contrast, the IL-18 levels immediately decreased after liver transplantation. Immunohistochemically, the number of CD68-positive Kupffer cells was significantly higher, and the size was larger in the livers of the patients than in the controls. The proliferation of CD68-positive cells was much more conspicuous in the liver specimens obtained during liver transplantation than in those at the time of HPE. CONCLUSIONS: Our findings showed elevation of serum IL-18 levels and activation of Kupffer cells in BA. IL-18 released from activated Kupffer cells might play an important role in the pathophysiology of the progressive inflammation and fibrosis in BA. Furthermore, IL-18 level may be related to the prognosis in patients with BA.     

Kupffer Cell Function in Ischemic and Nonischemic Livers After Hepatic Partial Ischemia/Reperfusion

Hepatic partial ischemic/reperfusion (I/R) injury, in which ischemic and nonischemic areas of the liver are likely to respond to each other after reperfusion, often occurs following hepatobiliary surgical procedures. Kupffer cells (KCs) are considered to play a major role in hepatic I/R injury. To study the activation of KCs in ischemic and nonischemic liver tissues following hepatic I/R, we investigated the superoxide generation and proinflammatory cytokine production of KCs in both liver parts in a rat model of partial hepatic I/R injury. KC superoxide generation in the ischemic and nonischemic lobes was upregulated 6 and 24 h after reperfusion, respectively, and then accelerated. The production of interleukin-1β (IL-1β) by KCs in the ischemic lobes increased during the early and late phases, 6 h and 48–72 h after reperfusion, respectively. A late increase in IL-1β production was also observed in the nonischemic lobes. Production of tumor necrosis factor-α (TNF-α) increased 6–24 h after reperfusion in both lobes. Upregulation of IL-1β mRNA in the ischemic lobes preceded the upregulation of TNF-α mRNA in both lobes. The hepatic partial I/R process results in activation of KCs in ischemic and nonischemic areas of the liver. The KCs are activated during the early phase after reperfusion in the ischemic areas, followed by activation in both the ischemic and nonischemic areas. This could be a cause of liver dysfunction after partial hepatic I/R during surgery. Received: September 9, 1999 / Accepted: September 26, 2000     

Kupffer cells participate in rejection following liver transplantation in the rat

Abstract Recognition of foreign antigens involves macrophages which release mediators such as immunoactive interleukins, and in the liver, the resident macrophages (Kupffer cells) are activated following transplantation. Therefore, we evaluated the hypothesis that Kupffer cells participate in the rejection reaction following transplantation. Orthotopic liver transplantation was performed between different syngenic rat strains. Livers from Lewis rats were stored in lactated Ringer's solution for 1 h to minimize cold ischemic injury and transplanted into PVG recipients. At 24 h postoperatively, transaminases (AST) were elevated to values around 2000 U/l, total bilirubin was increased to values around 20 μmol/l, and five of six rats died within 3 days. Macroscopic and histological examination showed large areas of necrosis without cellular infiltration, characteristic of rejection. When donor rats were treated with gadolinium chloride (GdCl₃, 10 mg/kg i.v. 24 h before storage of the liver) to inactivate the Kupffer cells, AST levels only rose to around 700 U/l, and the total bilirubin level was in the normal range (<4 μmol/l). Survival was improved significantly by GdCl₃, with five of seven rats surviving more than 1 month ( P < 0.05) and four of seven rats surviving for at least 100 days without immunosuppressive drug therapy. Rejection was not totally prevented, however, since the surviving rats had elevated AST and bilirubin levels, and cellular infiltration in portal areas along with proliferation of bile canaliculi was observed. These data are consistent with the hypothesis that Kupffer cells participate in mechanisms of early rejection following liver transplantation.