【作者】 杨落落；

【导师】 孟祥伟； 刘章旭；

【作者基本信息】 吉林大学 ， 内科学， 2014， 博士

【摘要】 肝脏是人体最大的器官。既往，肝脏的免疫功能曾被学者们忽视，近年来，越来越多的研究揭示了肝脏在免疫系统中的重要作用。肝脏免疫由固有免疫、适应性免疫及循环中的其它成份共同组成，其中固有免疫最为关键。枯否细胞（KC）是肝脏固有免疫的重要组成部分。KC生物学功能非常广泛，在肝脏的免疫防御中扮演着重要角色。KC有很强的吞噬能力，可以清除内毒素和其它来自循环中的外源性物质，也可以去除机体内的细胞碎片、病毒、细菌、凋亡细胞和一些肿瘤细胞等，在受损及感染后引起炎症反应。KC是专职抗原提呈细胞，可激活辅助T细胞参与触发适应性免疫反应。另外，KC也是机体最具分泌活性的细胞，可以分泌多种活性介质参与调节稳态、宿主抵抗、炎症等多种免疫反应。值得注意的是，KC还与很多肝脏疾病的发生、发展紧密相关。对乙酰氨基酚（APAP），即扑热息痛，是目前广为应用的解热镇痛药之一。在美国及欧洲国家，每年约有45%的急性肝衰竭患者由过量使用APAP引起。在我国，APAP的应用也十分广泛，除直接应用外，还是很多中、西药物的重要成份，市面上约有80%已批准抗感冒药（如：感康、泰诺、白加黑、百服宁和快克等）含有APAP。由于人们医疗知识缺乏，对药物认知不足，错误、过量服用或交叉服用含APAP成份药物的情况仍经常发生，导致患者严重肝、肾损伤甚至死亡的情况也不为少见。既往，APAP诱导的肝损伤机制的研究主要关注在毒性发挥早期阶段肝细胞内发生的生物化学代谢事件。然而，愈来愈多的证据显示固有免疫在促进和加剧APAP诱导的肝损伤中的重要作用。KC的重要作用已经在包括APAP在内的多种药物性肝损伤模型中得以证实。在炎性反应中，破损的肝细胞释放的细胞信号和死亡肝细胞释放的损伤相关分子模式均可激活KC。活化后的KC通过释放多种促炎性细胞因子、趋化因子、ROS和RNS进一步促进肝损伤及炎症反应的进展。另一方面，KC亦可以释放具有肝脏保护效果的细胞因子IL-4及抗炎性细胞因子IL-10。因此，KC在APAP诱导的肝毒性损伤中的作用是非常复杂的。近年来的研究中，学者们应用氯化钆和/或脂质体包裹的氯磷酸盐（CLO）使小鼠/大鼠肝脏内KC耗竭后观察APAP过量时肝损伤情况以评价KC在APAP诱导的肝毒性损伤中的作用。但是，实验结论未能统一，KC在APAP诱导的肝损伤中及损伤修复中的作用仍具争议。本研究的目的在于深入探讨KC在APAP诱导的肝毒性损伤的作用及可能原因。在本实验中，我们需建立KC缺失的APAP诱导的肝损伤动物模型。首先，经由小鼠尾静脉注射150ul脂质体包裹的氯膦酸盐溶液(CLO，5mg/ml)，CLO可被KC内吞并产生KC耗竭效应，对照组小鼠尾静脉注射等量脂质体溶液(LIP)。在LIP/CLO作用后的24h，即KC耗竭后再经腹腔注射毒性剂量的APAP（500mg/kg）以建立KC缺失的APAP诱导的肝损伤动物模型。血清ALT及肝脏病理切片将用于肝损伤程度的分析。肝非实质细胞及淋巴细胞被分离出来并通过流式细胞术（FACS）进行KC耗竭效果评价及细胞亚型分析。使用免疫组化F4/80抗体对肝脏组织切片中KC进行识别并进行KC耗竭效果评价。应用荧光定量聚合酶链反应（qPCR）法检测小鼠肝脏中细胞因子（TNF-α, IFN-γ, IL-6,IL-1-α, IL-1-β）及趋化因子（CCL2，CXCL1）含量。使用PCNA免疫组化染色及western blot法检测Cyclin D1蛋白表达以评价肝细胞增殖及损伤修复情况。为除外CLO及KC缺失对APAP药物代谢的影响，肝脏谷胱甘肽（GSH）浓度将通过Tietze法测得，CYP2E1蛋白质表达将通过western blot法进行测定。结果显示，KC在注射CLO24h后被基本耗竭。CLO对NK、NKT、T细胞及B细胞并没有产生影响。与预注射LIP的对照组相比，预注射CLO致KC耗竭的实验组动物在APAP所导致的肝毒性损伤的早期（6h）损伤程度较轻。其证据为，在给予APAP6h后相对较低的血清ALT（对照组为1598±257U/L，预注射CLO组为808±164U/L，p<0.05)及明显减少的小叶中央区坏死面积(35.5±7.0%和14.5±7.7%, p<0.05)。在APAP用药6h后与预注射LIP的对照组相比，预注射CLO致KC缺失组动物肝内浸润的炎性单核细胞(CD11b+，Ly6Chi)、招募巨噬细胞(CD11b+, F4/80+)和中性粒细胞(CD11b+，Ly6Ghi)较少，肝脏内趋化因子（CCL2，CXCL1）及细胞因子（TNF-α, IFN-γ, IL-6）mRNA的表达较低。但是，在给予APAP24h后，两组在血清ALT水平，肝脏病理水平上没有显著差异，且预注射CLO组小鼠肝脏内炎性单核细胞、招募巨噬细胞及中性粒细胞总数在APAP注射后24h后，达到了与对照组相近水平。由APAP诱导的肝损伤在APAP给药后72h基本恢复，APAP给药后48h及72h两组血清ALT及肝脏病理水平没有显著差异。另外，PCNA免疫组化染色结果及CyclinD1蛋白表达水平在给药后24h，48h及72h均无差异。两组间肝脏CYP2E1蛋白以及GSH消耗水平在APAP给药后组间均无差异。以上结果显示KC在APAP诱导肝毒性损伤早期具有促进损伤的作用，其作用机制可能与KC分泌具有促炎性作用的细胞因子及分泌趋化因子致招募炎性单核细胞、巨噬细胞及中性粒细胞增加有关。但是，KC的缺失未对肝损伤后期阶段及再生阶段的组织修复产生影响，可能是新招募的炎性单核细胞/巨噬细胞代替KC发挥了抗炎性及创伤修复功能。综上，我们的研究结果表明KC在APAP诱导的肝损伤早期具有损伤促进作用，为APAP诱导肝毒性损伤早期炎症控制提供新的思路。另外，提示了肝脏固有免疫与药物性肝损伤之间的密切联系，肝脏作为免疫器官值得我们继续探索。更多还原

【Abstract】 liver is the largest organ in the body. Previously, the immune function of theliver has been neglected by scholars, in recent years, more and more research revealsthe important role of the liver in the immune system. Liver immune system consists ofinnate immunity, adaptive immunity and other components in circulation, and theinnate immunity is the most important one. Kupffer cell (KC) is an importantcomponent of innate immunity of liver. KC’ biological functions are very extensive,and they play an important role in hepatic immune defense. KC has a strongphagocytic, which can remove endotoxin and exogenous substances from thecirculation, also be used to remove the body cells, cell debris, old virus, bacteria,apoptosis cells and some tumor cells. Initiating inflammation after damage andinfection. KC are professional antigen presenting cells, which can activate helper Tcells involved in triggering the adaptive immune response. In addition, KC is also themost secretory activity cells, can secrete several active mediators to modulatehomeostasis, host resistance, inflammatory reaction. Notably, KC also tightly relatedwith a lot of liver disease occurrence and development.Acetaminophen (APAP), also konw as paracetamol, is one of the widely usedanalgesic and antipyretic drug. In American and European countries, about45%acuteliver failure patients caused by APAP overdose each year. In China, APAP is widelyused, except direct application, it also as an important component in other drugs,about80%of the approved clodrex containing APAP. Due to the lack of knowledge of medical and pharmaceutical cognitive, overdose or cross use ingredients containingAPAP drugs are still frequent. Leading to liver and kidney serious damage and evendeath. Previously, the mechanism of liver injury induced by APAP mainly focus onthe biochemical and metabolic events occurring in early stage. However, more andmore evidence that the innate immune response plays an important role in promotingand aggravation of liver injury induced by APAP.Significant effect of KC has been demonstrated in a variety of drug-induced liverdamage model including APAP. In inflammation, cell signals and damage associatedmolecular patterns released by damaged liver cells and dead cell can activating KC.Activated KC can release proinflammatory cytokines, chemokines, ROS and RNS tofurther promote the hepatic injury and inflammation. On the other hand, KC can alsorelease cytokines IL-6and anti-inflammatory cytokine IL-10, which havehepatoprotective effect. Therefore, the role of KC in hepatotoxicity induced by APAPin is very complex. In recent year, for evaluating the role of KC in APAP inducedinjury, scholars use gadolinium chloride and/or lipo-clodronate (CLO) to depletedKC in mice/rats’ livers and observe liver injury after APAP challenge. However, theexperimental conclusion is not unified, KC in APAP induced liver injury and in therepair of the injured are still controversial. The purpose of this study is to explore KCfunction in APAP-induced liver injury.In this experiment, we have to set up KC-defected-APAP-induced-liver-injuryanimal model and then we i.p. injected500mg/kg of APAP dissolved in PBS toinduce liver injury. KC were depleted by i.v. injection of150ul of liposomalclodronate (clodrosome, CLO，5mg/ml)24h prior to APAP challenge; liposome wasused as vehicle control. SerumALT and histology were used for liver damage analysis.Hepatic non-parenchymal cells and leukocytes were isolated and assessed for cellsubtypes by flowcytometry (FACS). Immunohistochemistry(IHC) of liver sections for F4/80were performed to identify KC and confirm CLO effects.Fluorescent-Quantitation Polymerase Chain Reaction Assay for Cytokines（TNF-α,IFN-γ, IL-6, IL-1-α, IL-1-β） and chemokines（CCL2，CXCL1）. IHC for PCNA andwestern blot for Cyclin D1were used for detecting proliferating hepatocytes. To roleout the effect of CLO on metabolism of APAP, hepatic GSH concentrations wasmeasured using the method of Tietze, CYP2E1detected by western blot.The results display KC were effectively depleted at24h after CLO treatment,which was confirmed by hepatic F4/80IHC staining and FACS data. CLO treatmenthad no effect on the numbers of hepatic NK, NKT, T and B cells. Compared withAPAP-treated control mice, KC depletion by CLO pretreatment conferred protectionagainst APAP-induced early liver injury at6h evidenced by significantly reducedlevels of serum ALT (1598±257U/L in Controls vs.808±164U/L in CLO, p<0.05)and centrilobular necrosis area (35.5±7.0%vs.14.5±7.7%, p<0.05). The findingshowed decreased hepatic infiltration of inflammatory monocytes (CD11b+，Ly6Chi),recrument marophages (CD11b+, F4/80+) and neutrophils (CD11b+，Ly6Ghi),and reduced hepatic mRNA expressions for chemokines (CCL2, CXCL1) andcytokines (IL-6, TNF-a, IFN-γ) in CLO-treated mice at6h after APAP injected.However, at24h after APAP, there were no significant differences of liver injury interms of serum ALT and liver histology between these two groups. Interestingly, thenumbers of hepatic inflammatory monocytes, macrophages and neutrophils inCLO-treated mice reached to the similar levels of APAP-treated control mice at24hafter APAP. APAP-induced liver injury was resolved by72h in both groups and nodifferences of serum ALT and liver histology were observed at48h and72h despitethe continued absence of KC in the liver of CLO-treated mice. Furthermore, thePCNA positive hepatocytes in the liver displayed no differences at24h,48h and72hafter APAP overdose. Hepatic CYP2E1proteins and GSH depletions after APAP exhibited no differences between control and CLO-treated mice.These findings suggest that KC contribute to the early development ofAPAP-induced liver injury by producing inflammatory chemokines/cytokines torecruit inflammatory monocytes, macrophages and neutrophils, but their role in thelate phase of liver injury and regeneration could be replaced by newly recruitedinflammatory monocytes/macrophages. In a word, our results indicate that KCpromoting injury in the early phase of APAP overdose, which provides a newresearch thought of early inflammatory damage control in APAP-induced livertoxicity. In addition, point out the close links between innate immunity and liverdrug-induced liver injury, and liver as an immune organ worthy of our continuedexploration.更多还原