【作者】 黄汉飞；

【导师】 曾仲； 喻智勇；

【作者基本信息】 昆明医学院 ， 外科学， 2009， 硕士

【摘要】 自1963年Starzl施行首例临床肝移植手术以来,肝移植已成为当今治疗终末期肝病的唯一有效方法。然而,同其它大器官移植一样,供肝离体后的缺血再灌注损伤乃是面临的重大难题之一,常导致手术后供肝原发性无功能,被认为是肝移植失败的主要原因。移植肝脏经历冷缺血再灌注损伤后,会产生大量的氧自由基,促炎性细胞因子等加重肝脏的损伤,导致肝细胞坏死和器官功能衰竭。氧自由基是器官经历缺血再灌注后加重组织损伤的最早和最重要的成分之一,它们主要来源于细胞内黄嘌呤氧化酶的氧化作用,Kupffer细胞和多形核白细胞的缺氧刺激。多形核白细胞和Kupffer细胞产生的大量氧自由基导致内皮细胞损伤,最后使微血管完整性丧失和血流减少（无复流现象）。然而,到目前为止仍然没有一种有效的处理方法来预防肝脏缺血再灌注损伤。研究发现,血红素氧合酶-1（HO-1）对肝脏缺血再灌注损伤具有保护作用,但其具体的作用机制仍不十分清楚。已发现的血红素氧合酶系统有三种同工酶,HO-1为诱导型,又称为热休克蛋白32;HO-2为非诱导型,又称为结构型;HO-3与HO-2相关但含量较少。HO-1是一种血红素降解的催化酶和限速酶,可将血红素分解成一氧化碳（CO）、胆绿素（BV）和游离铁(Fe²⁺)。胆绿素被胆红素还原酶进一步还原为胆红素,游离铁参与细胞内代谢或结合为铁蛋白储存于体内,研究还发现CO能保护肝脏移植物缺血再灌注损伤并延长其冷保存时间。器官在多种应激情况下能诱导HO-1表达对组织器官起到保护作用,因此,HO-1对器官的缺血再灌注损伤有重要的抗炎性作用和临床应用潜能。Kupffer细胞是位于肝血窦内的巨噬细胞,寄居于肝血窦内皮细胞之间或之上,是体内固定型巨噬细胞中最大的群体,约占肝细胞总数的10—15%、全身定居巨噬细胞总数的80%左右。在缺血再灌注早期,Kupffer细胞被激活,产生形态学改变并突向窦状隙内导致血流减少。激活的Kupffer细胞释放多种活性介质,如:炎性细胞因子,活性氧类物质等。Kupffer细胞在肝脏缺血再灌注损伤早期发挥着重要的作用,有研究表明,抑制Kupffer细胞的激活能改善肝脏缺血再灌注损伤,因为激活的Kupffer细胞会导致一系列的炎性介质、生长因子和活性氧类物质的释放。通过单克隆抗体免疫组化结果发现HO-1主要分布在Kupffer细胞上,然而,HO-1在发挥保护肝脏冷缺血再灌注损伤时Kupffer细胞的发挥作用研究却很少。因此,本研究采用大鼠肝移植冷缺血再灌注模型,探讨HO-1保护肝脏缺血再灌注损伤与Kupffer细胞的作用,从而为HO-1保护缺血再灌注损伤作用的基础研究提供一定的理论基础和实验依据。方法:1.采用大鼠肝脏离体Ⅳ型胶原酶灌注,剪碎肝脏,37℃消化30分钟,低速离心去除肝细胞,Percoll不连续密度梯度离心和选择性贴壁的方法分离Kupffer细胞,细胞按2×10⁶/ml的密度用RPMI 1640培养液接种于培养板内,通过墨汁吞噬实验和ED2免疫细胞化学来鉴定Kupffer细胞。2.SPF级健康成年雄性SD大鼠,体重220～250g,被随机分为三组:①对照组:供肝不用任何药物处理;②锌原卟啉处理组:获取供肝24小时前腹腔注射HO-1抑制剂锌原卟啉;③钴原卟啉处理组:获取供肝24小时前腹腔注射HO-1诱导剂钴原卟啉。供肝使用4℃生理盐水灌注并冷保存90min后移植给同基因SD大鼠,门静脉开放6小时后获取标本。分离并培养Kupffer细胞,培养48小时后收集上清,提取Kupffer细胞总mRNA或蛋白质。观察移植前肝脏HO-1的表达水平,检测血清ALT和AST评价肝功能,组织病理观察肝细胞损伤程度,检测血浆和培养上清促炎性细胞因子IL-6和TNF-α的含量,通过RT-PCR和Western blot法测定Kupffer细胞CD14的表达水平。结果:1.分离的KCs的得率在细胞贴壁前2.1±0.3×10⁶/g肝脏、贴壁后1.5±0.1×10⁶/g肝脏,用0.4%台盼蓝染色,活率大于92%,ED2染色阳性的细胞达90%以上,分离的细胞培养后功能完整并延伸呈不规则型。2.获取大鼠供肝前使用锌原卟啉预处理后肝脏HO-1表达降低,钴原卟啉预处理后HO-1表达增加。钴原卟啉处理组大鼠血清转氨酶（ALT/AST）明显降低,而锌原卟啉处理组与对照组和钴原卟啉组相比增高。通过组织病理学观察发现锌原卟啉处理组肝细胞坏死严重、窦状隙充血肿胀,钴原卟啉处理组可见完整的肝小叶结构。检测血浆和细胞培养上清细胞因子（IL-6/TNF-α）的含量,锌原卟啉组细胞因子的含量明显增高,而钴原卟啉组明显降低。锌原卟啉处理后培养的Kupffer细胞HO-1表达明显降低,CD14mRNA和蛋白质水平表达上调,钴原卟啉处理后Kupffer细胞HO-1表达增高,CD14mRNA和蛋白质水平表达下调。结论:1.本实验建立的大鼠肝脏Kupffer细胞的分离培养方法简便、高效、稳定,培养的细胞具有良好的生物学性状,为进一步研究提供基础。2.HO-1具有保护大鼠移植肝冷缺血再灌注损伤的作用,能减轻肝脏损伤,抑制IL-6和TNF-α的产生,改善肝功能,抑制Kupffer细胞的激活可能是其作用机制之一。更多还原

【Abstract】 From the first report of Starzl,who performed clinical liver transplantation in 1963,orthotopic liver transplantation(OLT) is an effective treatment for end-stage liver diseases.Today,liver ischemia/reperfusion(I/R) injury is one of the confronted important problems and considered a major cause of graft injury,causing liver dysfunction and even failure post transplantation.The damage to the liver caused by I/R represents a continuum of processes that culminate in hepatocellular injury.These processes are triggered when liver is transiently deprived of oxygen and subsequent to reoxygenation,which inflicts direct tissue damage and initiates a cascade of deleterious cellular responses with concomitant release of oxygen-free radicals(OFR) and cytokines leading to cellular injury that culminates in the ultimate cell death and graft failure.OFR is one of the earliest and most important components of tissue injury after reperfusion of ischemic organs.The prime sources of OFR production in ischemic livers include cytosolic xanthine oxidase(XO),Kupffer cells(KCs) and adherent polymorphonuclear leukocytes(PMN).Endothelial cell(EC) damage results from free radicals produced from KCs and adherent PMN.Ultimately,this results in loss of microvascular integrity and reduction of blood flow(’no-reflow phenomenon’). However,at present there is no treatment available to prevent liver I/R injury.Studies have shown that heme oxygenase-1(HO-1) exerts a cytoprotective function in a number of liver I/R injury.However,exact mechanisms by which HO-1 induction may lead to cytoprotection during I/R injury in organ transplantation have not been fully clarified.Three HO isoforms have been identified:inducible HO-1, also known as heat shock protein 32;constitutively expressed HO-2;and a related but less well-characterized HO-3.HO-1 is the rate-limiting step in the oxidative degradation of heme into biliverdin,carbon monoxide(CO) and free iron.Biliverdin is reduced to bilirubin(BR) by bilirubin reductase,and the free iron used in intracellular metabolism or sequestered in ferritin.Studies have also reported that CO protects graft livers against cold I/R injury associate with prolonged cold preservation. HO-1 is induced in a variety of organs during diverse stress-related conditions and is thought to provide cytoprotection.Thus,HO-1 is attractive target for anti-inflammatory therapies and potential candidate responsible for organs I/R injury.Kupffer cells,the resident macrophages of the liver,are the largest population of tissue macrophages and predominantly distributed in the lumen of hepatic sinusoids. KCs account for approximately 10～15%of all liver cells in number and for 80%of resident macrophages in the whole body.During the initial stages of reperfusion KCs are activated,producing morphologic changes that cause them to protrude into the sinusoids,contributing to the reduction of blood flow within the sinusoidal lumen. Activated KCs release a large amount of biologically active mediators such as proinflammatory and reactive oxygen species(ROS).Multiple lines of evidence have suggested that Kupffer cells are critical to the onset of liver I/R injury.Studies have shown that liver I/R injury can be attenuated by the suppression of KCs.Because activation of KCs results in the release of an array of inflammatory mediators,growth factors and ROS.Immunochemical studies with specific monoclonal antibodies have revealed that HO-1 express principally in Kupffer cells.However,little is known about the roles of KCs in HO-1 protecting liver cold I/R injury.In the present study,we investigated the roles of KCs and protective effect of HO-1 in liver cold I/R injury model.We’ll provide rationally experimental evidence accordance with this study for further research.Methods: 1.Ex vivo rat liver tissue was perfused with collagenaseⅣand diced into pieces. The diced tissue was digested for 30 min at 37℃and following centrifuged to remove hepatocytes.Percoll density gradient centrifugation and selective adherence were combined.The cells were seeded onto tissue culture plates at a density of 2×10~6/ml and cultured in RPMI 1640 medium.Phagocytosis of ink and ED2 immunocytochemistry were used for cell identification.2.Male Sprague-Dawley(S-D) Rats weighing 220-250g were used and divided randomly into three groups:①Control group:no drugs were applied;②ZnPP treatment group:donors received ZnPP,an HO-1 inhibitor(i.p.) 24h prior to harvest;③CoPP treatment group:donors received CoPP,an HO-1 inducer(i.p.) 24h prior to harvest.All liver grafts were harvested and stored with saline solution for 90min at 4℃,and orthotopically transplanted into syngeneic S-D recipients.Separate groups of rats were killed at 6h after their vessels were unclamped,and liver samples were collected for further analysis.Kupffer cells were isolated from integral left liver tissue obtained from fresh specimens and were incubated for 48h.The supematant from Kupffer cells culture was collected,and total RNA or proteins were extracted from Kupffer cells.HO-1 expression in livers pretransplant was assessed,and the hepatocellular function(ALT,AST) was analyzed.The hepatocellular damage was evaluated by liver histology.The IL-6/TNF-αlevels of plasma and supematant were detected.CD14 mRNA expression levels of KCs were examined by RT-PCR and Western blot.Results:1.The average cell yield per gram of liver before plastic adherence was 2.1±0.3×10~6,and 1.5±0.1×10~6 following plastic adherence.The viability of the KCs after isolation was＞92%as determined by trypan blue exclusion.The purity of KCs identified by ED2 was＞90%.Cultures of isolated KCs were functionally intact and expanded irregularly.2.Livers harvested from donors that were pretreated with CoPP significantly up-regulated HO-1 mRNA expression levels as compared with Control or the ZnPP group.ZnPP treatment decreased HO-1 mRNA as compared with Control.Group pretreatment with CoPP significantly decreased Serum ALT/AST levels.Serum AST/AST levels were also significantly increased in the ZnPP treatment group as compared with both the Control and the CoPP group.The hepatocellular damage was evaluated by liver histology.Livers in ZnPP group showed a more severe hepatocyte necrosis,sinusoidal congestion and ballooning.In contrast,livers in CoPP group revealed almost complete preservation of lobular architecture.Plasma and supernatant cytokines(IL-6/TNF-α) concentrations were detected.ZnPP treatment significantly increased after reperfusion.However,the IL-6/TNF-αlevels in CoPP pretreated group were significantly lower,when compared to the Control group and ZnPP group.HO-1 mRNA expression levels of Kupffer cells with ZnPP pretreated were significantly down-regulated,and those with CoPP pretreated were up-regulated.A prominent increase of CD 14 mRNA and protein levels in Kupffer cells was detected in the ZnPP group as compared with the Control group or the CoPP group.Conclusions:1.The method of isolating and culturing Kupffer cells in this study is convenient, efficient and stable.The biological characters of the cultured Kupffer cells were retained,and the cells can be used to further study.2.HO-1 protects liver against I/R injury in rat liver transplantation,ameliorating hepatic damage,suppressing cytokines IL-6 and TNF-αrelease and improving liver function.It is possible that HO-1 inhibiting Kupffer cells activation is one of the mechanisms.更多还原