【作者】 姚景宏；

【导师】 贺永文；

【作者基本信息】 华中科技大学 ， 内科学， 2007， 博士

【摘要】 肝细胞损伤是临床各类肝脏疾病的最常见病理改变之一,引起肝细胞损伤的诱发因素包括病毒感染、酒精和化学毒性物质等,其分子机制与自由基生成、脂质过氧化反应、钙超载以及单核/巨噬细胞功能状态等密切相关。各类机制之间相互联系、相互促进,共同作用引起肝细胞凋亡、变性及坏死。目前认为,肝脏不仅是人体的最大消化腺,也是重要的免疫器官。在肝脏中有大量的免疫细胞,如树突状细胞、Kupffer细胞等,广泛参与人体的免疫反应,免疫细胞功能失控必然导致肝细胞损伤。机体的免疫反应通常可分为天然免疫和获得性免疫,其中天然免疫是机体抵御病原入侵的第一道防线,同时能调节获得性免疫的方向和强度。肝组织中的Kupffer细胞、肝窦内皮细胞和树突状细胞均可参与天然免疫,其中Kupffer细胞数量巨大,功能最强,是人体单核/巨噬细胞系统的重要组成部分。因此,我们以单核/巨噬细胞为主要研究对象,探讨其介导的天然免疫在肝细胞损伤中的作用机制。本研究共分五个部分,以单核/巨噬细胞为主要研究对象,通过研究单核/巨噬细胞的TLR4受体及其信号通路变化,揭示单核/巨噬细胞介导的天然免疫反应在肝细胞损伤中的作用。首先,为了研究病毒性肝炎患者内毒素血症及外周血单核细胞内TLR4 mRNA的表达的关系,我们收集各型病毒性肝炎患者及正常人外周静脉血,采用鲎试剂法测定血浆脂多糖(LPS)浓度,用RT-PCR法检测外周血单核细胞TLR4 mRNA的表达。结果显示:肝炎患者血浆LPS浓度均不同程度的高于对照组,以重型肝炎组升高最显著(P<0.05);重型肝炎组单核细胞TLR4 mRNA的表达升高也最明显(P<0.05),而其他各型肝炎与对照组比较差异不明显(P>0.05)。提示各型肝炎均存在内毒素血症,以重型肝炎最为严重,且重型肝炎外周血单核细胞内TLR4 mRNA的表达水平最高,可能与肝细胞损伤相关。为了研究在急性肝损伤时肝组织TLR4的表达变化及其与肝细胞损伤的关系,我们用D-Gal/LPS制作大鼠急性肝损伤模型,HE染色观察肝组织病理变化程度,并于不同时间点检测肝功能指标,免疫组化观察肝组织TLR4的表达,TUNEL法观察肝脏细胞凋亡情况。同时运用NF-κB特异性抑制剂二硫氨基甲酸酞吡咯烷(pyrrolidine dithiocarbamate,PDTC)阻断TLR4的下游信号通路,研究抑制TLR4信号转导对肝损伤的影响。结果显示:急性肝损伤模型鼠在4~24h ALT、AST等生化指标异常升高,肝组织TLR4表达增强,凋亡的肝脏细胞数随时间的延长逐渐增多;PDTC干预后可明显减轻肝组织病理改变,与模型组相比,TLR4表达较弱,肝脏细胞凋亡也显著减少,提示TLR4可通过激活NF-κB途径及其下游通路在急性肝损伤的发生发展中发挥重要作用。在肝组织中,Kupffer细胞表达的TLR4是结合LPS的主要受体,但肝细胞、肝窦内皮细胞也表达少量的TLR4,为了进一步明确Kupffer细胞表达的TLR4在急性肝损伤中发挥的作用,我们在大鼠急性肝损伤模型基础上,运用GdCl3对肝脏Kupffer细胞有特异抑制作用,通过免疫组化检测ED1表达,ED1是Kupffer细胞特异性分子标记蛋白,判断GdCl3对Kupffer细胞的抑制程度,并检测不同实验组动物血清ALT、TNFα、IL-1β的水平,Western blot分析肝组织TLR4的表达。在GdCl3组大鼠的肝组织中观察到,与对照组相比ED1阳性细胞明显减少(P<0.05),其门静脉血中ALT、TNFα、IL-1β的含量无明显变化(P>0.05)。而比较GdCl3+LPS组与LPS组的肝组织病理改变发现,GdCl3预处理明显减轻了D-Gal/LPS诱导的肝组织病变(肝细胞变性、坏死)程度,同时抑制了D-Gal/LPS诱导的炎前细胞因子TNFα、IL-1β升高(P<0.05),说明GdCl3抑制了Kupffer细胞的功能,进而影响肝组织TLR4的表达,并减少炎前细胞因子的释放,进而减轻LPS对肝细胞的损伤。此部分进一步证实了Kupffer细胞及其表达的TLR4在急性肝损伤中的重要作用。LPS激活Kupffer细胞不仅能诱导其产生IL-1、TNFα等炎前细胞因子,还可产生大量的趋化因子,募集外周血免疫细胞进入肝组织。Kupffer细胞是肝脏中唯一表达、合成5-脂氧合酶(5-lipoxygenase,5-LO)的细胞,而5-LO是白三烯类代谢物生成的关键酶,能催化花生四烯酸生成5-羟基廿碳四烯酸(5-HETE)、白三烯(Leukotrienes, LTs)等物质,其中LTs则是中性粒细胞的最强趋化因子之一。为了研究LPS激活Kupffer细胞TLR4后,细胞合成、释放炎性介质的变化,我们用原位酶灌注,密度梯度离心等方法,分离、纯化培养原代Kupffer细胞,LPS刺激后,于12h、24h、48h收集不同实验组细胞培养上清液,硝酸还原酶法测定NO水平,ELISA法检测TNFα含量,半定量RT-PCR法检测细胞5-LO mRNA的表达。同时观察了免疫调制激素-生长抑素(somatostatin,SST)对LPS作用的影响。研究结果显示:分离培养的Kupffer细胞表达低水平的NO、TNFα和5-LO mRNA。LPS刺激后各时间点NO、TNFα和5-LO mRNA含量均明显高于对照组(P<0.05)。SST干预后各时间点NO、TNFα和5-LO mRNA含量低于LPS刺激组(P<0.05),而NO、TNFα含量在各时间点均高于对照组(P<0.05),5-LO mRNA表达在12h、24h时间点高于对照组(P<0.05),而在48h时间点与对照组相比,差异无统计学意义(P>0.05)。SST能抑制LPS诱导的Kupffer细胞NO,TNFα和5-LO mRNA的表达。以上结果提示,LPS可通过TLR4激活Kupffer细胞产生大量的炎性介质,而SST则可通过抑制LPS对Kupffer细胞的作用,使其NO、TNFα和5-LO mRNA的表达减少,减轻炎性细胞浸润,从而发挥肝细胞保护作用。为了进一步研究内毒素血症时,Kupffer细胞激活对肝细胞代谢和增殖的直接影响,我们收集了Kupffer细胞的条件培养基(Kupffer cell conditioned medium,KCCM),作用于分离、培养的原代肝细胞,通过检测肝细胞培养基ALT、AST、乳酸脱氢酶(LDH)的含量,四甲基偶氮唑蓝(MTT)法检测KCCM对原代肝细胞活性的影响,观察KCCM对肝细胞的损伤情况。结果显示,KCCM作用于肝细胞2h后,肝细胞培养基中ALT、AST及LDH含量均明显升高(P<0.05),在2~24h内随时间延长ALT、AST及LDH含量均逐渐增加,呈明显的时间-效应关系;MTT结果显示,KCCM作用后12h、24h时间点对肝细胞具有明显损伤作用(P<0.05)。以上结果提示:LPS诱导Kupffer细胞释放的可溶性因子可直接对肝细胞造成损伤。小结:各型肝炎均存在内毒素血症,以重型肝炎最为显著,且重型肝炎外周血单核细胞内TLR4 mRNA的表达水平最高。Kupffer细胞及其表达的TLR4在急性肝损伤的发生发展中发挥一定作用。生长抑素能抑制LPS诱导的Kupffer细胞NO、TNFα和5-LO mRNA的表达,对肝细胞具有保护作用,在内毒素血症治疗方面具有一定的应用前景。内毒素诱导Kupffer细胞释放的可溶性因子可直接对肝细胞造成损伤。更多还原

【Abstract】 Hepatocyte injury was the most common pathological changes of clinical liver diseases. The inductive factors included virus infection, alcohol, chemical toxin, and so on. Its molecular mechanisms involved the production of free radical, lipoperoxide, calcium over-loaded and functional changes of monocytes/macrophages. All those factors are reciprocal, and act synergically, which lead to apoptosis, degeneration and necrosis of hepatocyte. Recently, it is believed that the liver is not only the biggest digestant gland in human body, but also one of the most important immune organs. The liver contains a large population of immune cells, such as dendritic cells and Kupffer cells, and takes part in immune response of human body. The uncontrolled functions of immune cells consequentially cause hepatocyte injury. The immune response can be man-madely divided into two parts, innate immunity and adaptive immunity. The innate immunity is the first line of defense, which can regulate the direction and intensity of adaptive immunity. The Kupffer cells, liver sinusoidal endothelial cells and dendrite cells in liver all can take part in the innate immunity, among which Kupffer cells are a member of the monocytes/ macrophages, the most numerous and have the strongest immune function. Therefore, our studies focused on the monocytes/macrophages to explore the mechanism of hepatocyte injury mediated their innate immunity.This dissertation included five sections. The innate immune functions of monocytes/macrophages were principally investigated through studying the TLR4 expression and its changes of signal transduction. The results indicated the innate immunity mediated by monocytes/macrophages play an important role in hepatocyte injury.Firstly, in order to study the relationship between the changes of TLR4 mRNA expression of peripheral blood monocytes and endotoxemia in patients of viral hepatitis, peripheral venous blood was obtained from hepatitis patients and health volunteers. The LPS concentration of plasma was detected by limulus lysate test. RT-PCR was used to detect the expressive change of TLR4 mRNA. The results showed that in the patients, LPS concentration of plasma was elevated in different degrees. In severe hepatitis group, the elevation of the LPS concentration was higher than that of other hepatitis group (P<0.05). The expression of the TLR4 mRNA in severe group was increased significantly compared with those of control group (P<0.05). However, in other hepatitis group, TLR4 mRNA expression was not significantly changed compared with those of control group ( P>0.05). The results indicated that endotoxemia was present in various types of the hepatitis patients in different degree, which was more prominent in severe hepatitis group. The expressions of TLR4 mRNA in peripheral blood monocytes in severe hepatitis patients were the highest, which might be correlated closely with hepatocyte injury.The purpose of second part is to study the relationship between the expressive change of TLR4 in the liver and hepatocyte injury in animal models of acute liver injury. The chemical toxins, D-Gal and LPS, were used to induce acute liver injury in rats. The degree of pathological changes in liver tissues was observed by HE staining. Serum transaminase (ALT and AST) and TLR4 in the liver and the apoptosis of liver cells were determined at different time points. We used specific inhibitor of NF-κB–PDTC to inhibit TLR4 downstream signal transduction, to study the influence of inhibition of TLR4 on liver injury. The results showed the serum transaminase level was elevated at 4, 8, 12, 24 hour time point after injection of D-Gal/LPS (P<0.05). The expression of TLR4 and the number of apoptotic cells in the liver increased gradually, which was higher than that of the normal control group (P<0.05). However, after treatment with PDTC, serum transaminase level, the expression of TLR4 and the number of apoptotic cells in the liver were lower than those of the model group (P<0.05). It indicated that TLR4 play an important role in acute liver injury through activating NF-κB pathway.The TLR4 expression of Kupffer cells is the main receptor which combined LPS in the liver. However, hepatocytes and liver sinusoidal endothelial cells were also expressed TLR4. To elucidate the expression of TLR4 in Kupffer cells play a role in acute liver injury, the rats were treated by Gadolinium Chloride (GdCl3) to specifically inhibit the function of Kupffer cells before the D-Gal/LPS administration, which did not affect the hepatocyte and other nonparenchymal cells. ED1 expression in liver tissues was observed by immunohistochemistry to identify the degree of inhibiting of Kupffer cells. Serum ALT, TNFαand IL-1βlevels were measured in different groups, and the expression of TLR4 in the liver was detected by Western blot. The number of ED1 positive cells in liver tissues of GdCl3 group were lower significantly than that of control group (P<0.05).Portal venous blood ALT, TNFαand IL-1βlevels was not elevated in rats receiving GdCl3 alone. Moreover, the degree of degeneration and necrosis of hepatocyte in D-Gal/LPS group was lower than that of LPS group (P<0.05). Serum ALT concentration was elevated in the D-Gal/LPS treated rats, and this increase was reduced with GdCl3 pretreatment. Similar results were observed with serum TNFαand IL-1βlevels (P<0.05). The expression of TLR4 in GdCl3 +LPS group was significantly lower than that of LPS group (P<0.05), but had no statistically significant difference compare with control group (P>0.05). Taken together, all results indicated that the TLR4 expression was decreased through inhibiting the activation of Kupffer cells stimulated by D-Gal/LPS in liver tissues, and Kupffer cells and its TLR4 play an important role in acute liver injury.The Kupffer cells activated by LPS can product not only the pro-inflammatory cytokines, such as IL-1 and TNFα,but also some kinds of chemotactic factors resulting in the rapid recruitment of immune cells to the liver. Expression of 5-lipoxygenase (5-LO) in the liver is basically restricted to Kupffer cells. 5-LO is the key enzyme that can catalyze oxygenation of arachidonic acid to 5(s)-hydroperoxy-6-trans-8, 11, 14-9-eicosatetraenoic (5-HPETE) and leukotrienes (LTs), which is one of the most strong chemokine of neutrophils. It is worthy of researching the synthesis and release of inflammatory mediators after TLR4 activation of Kupffer cells stimulating by LPS. The purified Kupffer cells were isolated from liver of Sprague-Dawley rats by in situ perfusion with pronase and collagenase and density gradient centrifugation with Percoll reagent. Then, those cells were treated by LPS, the production of TNFαwas assessed in culture supernatants by ELISA and that of nitric oxide (NO) by nitrate reducase method. 5-LO mRNA expression was assessed by semiquantitative RT-PCR. At the same time, Somatostatin (SST) was used to evaluate the immunity modulator’s action on the functions of Kupffer cells. The results showed that vehicle-stimulated Kupffer cells produced a basal amount of No, TNFαand expressed 5-LO mRNA. Kupffer cells stimulated by LPS secreted significantly increased amounts of NO and TNFαand 5-LO mRNA (P<0.05). The secretions of NO and TNFαand 5-LO mRNA induced by LPS were inhibited by SST. The amount of NO and TNFαwas higher than control group at each time point, however 5-LO mRNA expression was still higher than that of control group at 12 and 24h (P<0.05), but had no statistically significant difference compare with control group at 48h (P>0.05). The data presented in this part study strongly supported the views that Kupffer cells can produce a great lot of inflammatory mediators induced by LPS and SST can modulates the Kupffer cells function through inhibiting No, TNFαproduction and 5-LO mRNA expression, which alleviate inflammatory filtration and in turn play roles in hepatocyte protection.Finally, in order to explore the direct effects of Kupffer cells activated by LPS on the metabolism and proliferation of rat hepatocytes in endotoxemia, the Kupffer cells were stimulated by LPS and Kupffer cell conditioned medium (KCCM) were collected and administrated to hepatocytes. After hepatocytes exposured to KCCM, transaminase (ALT and AST) and lactate dehydrogenate (LDH) were measured in the supernatants in different time points. Meanwhile, MTT colori metric assay was used to detect the action of KCCM on hepatocytes viability. The results showed that the concentrations of ALT, AST and LDH were gradually increased 2h after exposure (P<0.05). There was remarkable time-dependent relationship between KCCM and release of transaminase and LDH in the supernatants of rat primary cultured hepatocytes from 2h to 24h. The results of MTT suggested that hepatocytes was obviously injured at 12h and 24h(P<0.05). The above results indicated that the soluble factors released by Kupffer cells, which were activated by LPS, play a role in hepatocyte injury directly.Summary:Endotoxemia was present in various hepatitis groups, which was more prominent in severe hepatitis group. The expression of TLR4 mRNA in peripheral blood monocytes in severe hepatitis patients was the highest, which might be closely correlated with liver injury. Kupffer cells and the expression of TLR4 might be involved in acute liver injury. SST inhibits NO, TNFαproduction and 5-LO mRNA expression by LPS-stlimulated Kupffer cells which has protective effect on hepatocytes. The soluble factors released by Kupffer cells, which were activated by LPS, play a role in hepatocyte injury directly.更多还原