Suppression of autophagy sensitizes Kupffer cells to endotoxin

Aim: Recent evidence suggests that protein degradation system autophagy is implicated in a component of innate immunity. We report here that suppression of autophagy in Kupffer cells due to hepatic steatosis enhances an inflammatory response to endotoxin. Methods: Kupffer cells were isolated from C57BL/6J mice fed chow diet (control) or high‐fat diet (HFD) for 12 weeks, liver‐specific autophagy‐deficient mice (Atg7^F/F:Mx1‐Cre) and wild‐type mice (Atg7^F/F). Kupffer cells were incubated with 100 ng/mL lipopolysaccharide (LPS). The concentration of tumor necrosis factor (TNF)‐α in media was measured by enzyme‐linked immunoassay. Expression of Toll‐like receptor (TLR)4, IκB kinase (IKK)‐α/β, p38, p62 and LC3 in Kupffer cells was evaluated by western blot analysis. Results: Incubation with LPS increased LC3‐II expression of Kupffer cells from control mice; however, an increase in LC3‐II expression due to LPS was suppressed in Kupffer cells from HFD mice. Moreover, both p62 expression and TNF‐α production in Kupffer cells from HFD mice was higher than control mice. On the other hand, LPS exposure increased TNF‐α production from autophagy‐deficient Kupffer cells more than wild type. There was no significant difference in expression of TLR4 between wild and autophagy‐deficient Kupffer cells. Nevertheless, activation of p38 or IKK in Kupffer cells due to LPS was augmented by autophagy deficiency. The addition of the p38 inhibitor SB203580 attenuated TNF‐α production in both wild and autophagy‐deficient Kupffer cells. Conclusion: These results suggest that suppression of autophagy observed in Kupffer cells from steatotic liver sensitizes to endotoxin. In conclusion, suppression of autophagy may play a pivotal role on progression of NAFLD.     

An in vitro model for the study of phagocytosis of damaged hepatocytes by rat Kupffer cells

Abstract: Aims/Background: One function of Kupffer cells is the phagocytosis of nonviable hepatocytes. Our aims were to develop a model for phagocytosis of damaged hepatocytes by rat Kupffer cells in vitro, and to characterise prostaglandin E₂ (PGE₂), prostacyclin (PGI), and tumour necrosis factor-α (TNF) production in this model. Methods: Kupffer cells were incubated alone or with damaged hepatocytes for up to 18 h, then washed and cultured for up to 66 h. To compare mediator responses produced during inert particle phagocytosis, Kupffer cells were also incubated with latex beads. Results: Phagocytic uptake of hepatocyte debris was confirmed in at least 50% of Kupffer cells. A dissociation between TNF and PGI responses was found for both latex beads and damaged hepatocytes, such that a TNF secretory response was not triggered by either stimulus whereas PGI production was increased for both. Although phagocytosis of beads increased PGE₂ production, phagocytosis of hepatocytes did not. Conclusions: Phagocytosis of damaged hepatocytes by Kupffer cells results in the production of PGI but not PGE₂ or TNF.     

库普弗细胞功能状态对肠缺血再灌注小鼠肝细胞凋亡和血清肿瘤坏死因子的影响

目的 研究库普弗细胞功能状态对肠缺血再灌注小鼠肝细胞凋亡和血清肿瘤坏死因子的影响。方法 封闭和不封闭BALB／c小鼠库普弗细胞(从尾静脉注射GdCl3或生理盐水27ml／kg体重)48h后，夹闭肠系膜上动脉1h后松夹，复制肠缺血再灌注模型。运用流式细胞术和酶联免疫法，分别检测缺血前、缺血60min、再灌注30min、60min肝细胞凋亡情况和血清肿瘤坏死因子的变化。结果 结果表明，肠缺血60min、再灌注30min、60min时，肝细胞凋亡数增多，血清肿瘤坏死因子水平逐渐升高；封闭库普弗细胞后，肝细胞凋亡数增多更显著，血清肿瘤坏死因子水平在同时间点上无显著差异。结论 库普弗细胞功能状态的变化对肠缺血再灌注时肝损伤有重要影响。     

Role of Kupffer cells in the outgrowth of colorectal cancer liver metastases

Colorectal cancer is one of the commonest malignancies in the developed world. The liver constitutes the main host organ for its distant metastases which, when present, augur a bad prognosis for the disease. Kupffer cells (KCs) are macrophages that constantly reside within the liver and form an effective first line defence against multiple harmful agents which reach the hepatic sinusoids via the portal circulation. KCs remove chemical compounds and dead or damaged cells, eliminate bacteria and protect against invading tumour cells. They may play a crucial tumouricidal role, exerting cytotoxic and cytostatic functions through the release of multiple cytokines and chemokines. Subsequently, colorectal metastasising cells are destroyed either by KC-performed phagocytosis or via the stimulation of other immune cells which migrate into the sinusoids and act accordingly. On the contrary, KC products, including cytokines, growth factors and matrix-degrading enzymes may promote liver metastasis, supporting tumour cell extravasation, motility and invasion. Current research aims to exploit the antineoplastic properties of KCs in new therapeutic approaches of colorectal cancer liver metastasis. Numerous agents, such as the granulocyte macrophage-colony stimulating factor, interferon gamma, muramyl peptide analogues and various antibody based treatments, have been tested in experimental models with promising results. Future trials may investigate their use in everyday clinical practice and compare their therapeutic value with current treatment of the disease.     

Characterization of macrophage function in Mycobacterium lepraemurium-infected mice: sensitivity of mice to endotoxin and release of mediators and lysosomal enzymes after endotoxin treatment

Mycobacterium lepraemurium (MLM) infection increases the sensitivity of mice to lipopolysaccharide (LPS) as do infections with other intracellular parasites. Tumour necrosis factor (TNF), lymphocyte activating factor (LAF) and increased levels of various lysosomal and cytoplasmic enzymes were found in serum samples taken 2 h after intravenous injection of a small dose of LPS suggesting that damage to a variety of cell types, including macrophages, had occurred. Sera from moribund MLM-infected mice not injected with LPS also demonstrated significant levels of TNF compared with controls. Intravenous injections of silica into leprous mice also led to increased levels of serum lysosomal and cytoplasmic enzymes but did not give rise to a significant amount of TNF or LAF. Moreover, in contrast to LPS treatment, the injection of silica did not lead to the death of leprous mice. These findings suggest that the phagocytic cells of the infected animals did not contribute to the production of these mediators after LPS challenge. Rather, the non-phagocytic granuloma macrophages or other unidentified cell types seemed to provide the main source of the monokines TNF and LAF in vivo in the present model. These mediators may have important implications for the immunopathology of MLM infection.     

鲜生地对肝损伤模型大鼠枯否细胞功能的影响

目的探讨鲜生地在大鼠肝损伤时对肝脏枯否细胞功能的影响。方法取健康成年大鼠64只,随机分成4组:假手术组,肝损伤组,肝损伤+鲜生地组和肝损伤+乳果糖组。前两组以生理盐水灌胃,后两组分别以鲜生地和乳果糖灌胃。观察大鼠血浆内毒素(ET)、肿瘤坏死因子-α(TNF-α)、白介素-1(IL-1)、谷丙转氨酶(ALT)以及枯否细胞表面CD68蛋白表达。结果与肝损伤组比较,鲜生地组和乳果糖组血浆ET、TNF-α、IL-1、ALT明显降低(P<0.05),CD68蛋白表达减少。结论鲜生地具有抑制肝脏枯否细胞吞噬及分泌作用,减少炎症因子释放,从而减轻肝细胞损伤。     

CD14 expression on Kupffer cells during the course of carbon tetrachloride‐mediated liver injury

OBJECTIVE: To investigate the expression of CD14 on Kupffer cells during the course of carbon tetrachloride (CCl₄)‐mediated liver injury and its role in the activation of Kupffer cells. METHODS: Rats were administered CCl₄ twice weekly for up to 8 weeks. Kupffer cells were isolated from normal and CCl₄‐treated rats by the combined ‘collagenase‐pronase’ perfusion method, discontinuous density gradient centrifugation. On the day after isolation, the cells were incubated with RPMI‐1640 containing varying doses of lipopolysaccharide (LPS) for 6 h. Supernatants were then collected for measuring the concentration of tumor necrosis factor‐alpha (TNF‐α) by enzyme‐linked immunosorbent assay (ELISA). The expression of CD14 mRNA on Kupffer cells were determined by RT‐PCR. The plasma concentrations of endotoxin were determined by chromogenic substrate Limulus amebocyte lysate assay. RESULTS: Basic TNF‐α production of Kupffer cells isolated from CCl₄‐treated rats at 4 and 6 weeks was significantly higher than that of normal (P < 0.05). Following LPS stimulation the production of TNF‐α was markedly increased in Kupffer cells from the 2‐, 4‐ and 6‐week treatment groups (P < 0.05). Moreover, LPS‐induced TNF‐α production was dose‐dependent. CD14 mRNA expression on Kupffer cells isolated from CCl₄‐treated rats was elevated following 2 weeks of CCl₄ administration and the maximum elevation occurred at 6 weeks. Gene expression was decreased in Kupffer cells after 8 weeks of CCl₄ treatment. CCl₄ administration elicited extensive changes in liver morphology, including steatosis, inflammation and necrosis. The plasma concentrations of endotoxin of CCl₄ ‐ treated rats were increased during the time of liver injury. CONCLUSION: Up‐regulation of CD14 expression in Kupffer cells during CCl₄‐mediated chronic liver injury indicates cell activation and that they are more sensitive to LPS stimulation. Kupffer cells are critical effector cells in the early stage of liver injury.     

Dexamethasone attenuates lipopolysaccharide-induced liver injury by downregulating glucocorticoid-induced tumor necrosis factor receptor ligand in Kupffer cells

Aim: Glucocorticoid‐induced tumor necrosis factor receptor ligand (GITRL) plays pro‐inflammatory roles in immune response. Thus, our aim was to assess if dexamethasone attenuates lipopolysaccharide (LPS)‐induced liver injury by affecting GITRL in Kupffer cells (KC). Methods: A BALB/c mouse model of liver injury was established by i.p. injecting with LPS (10 mg/kg) co‐treated with or without dexamethasone (3 mg/kg). Blood and liver samples were obtained for analysis of liver morphology, GITRL expression, hepatocellular function and cytokine levels at 24 h after injection. KC were isolated and challenged by LPS (1 µg/mL), with or without dexamethasone (10 µM) co‐treatment, or with GITRL siRNA pre‐transfection. The GITRL expression and cytokine levels were assayed at 24 h after challenge. Results: Dexamethasone treatment significantly improved the survival rate of endotoxemic mice (P < 0.05), whereas serum alanine aminotransferase, aspartate aminotransferase, tumor necrosis factor (TNF)‐α, interleukin (IL)‐6 and γ‐interferon levels were significantly decreased (P < 0.05, respectively). Concurrently, LPS‐induced hepatic tissue injury was attenuated as indicated by morphological analysis; and expression of GITRL in liver tissue and KC was downregulated (P < 0.05). Consistent with these in vivo experiments, inhibited expression of GITRL, TNF‐α and IL‐6 caused by dexamethasone treatment were also observed in LPS‐stimulated KC. The GITRL, TNF‐α and IL‐6 expression was also significantly inhibited by GITRL gene silencing. Conclusion: The TNF‐α and IL‐6 expression of LPS‐stimulated KC was inhibited by GITRL gene silencing. Dexamethasone attenuates LPS‐induced liver injury, at least proportionately, by downregulating GITRL in KC.     

Monokine gene expression in normal human liver: selective involvement of the portal compartment

Abstract: Monokines play a major role in the regulation of hepatocyte functions. To document a possible in situ production of these mediators under physiological conditions, expression of IL-1β and of IL-6 genes was analyzed by in situ hybridization in four histologically normal human livers. We detected cells containing IL-1β mRNA or IL-6 mRNA in all cases. Cells expressing either the IL-1β gene or the IL-6 gene were found with equal frequency and were similarly distributed. Although present in all liver compartments, they were selectively enriched in portal areas, in which they were detected both in endothelial positions and in perivascular connective tissues. Few positive cells were observed in hepatic lobules, most of them being located in the walls of centrolobular veins, in an endothelial position. Subcapsular cells were also shown to express monokine genes. The location of positive cells and their pattern of labelling suggested that macrophages, fibroblasts and endothelial cells were the main cell populations expressing monokine genes. In contrast, Kupffer cells, biliary epithelial cells and hepatocytes did not express monokine genes. No marker of immune activation other than monokine gene expression was detected in these histologically normal livers. The expression of the IL-1β gene and of the IL-6 gene may be induced by gut-derived LPS, and could play a role in the modulation of hepatocyte function in normal liver.     

Translocation of platelets into Disse spaces and their entry into hepatocytes in response to lipopolysaccharides, interleukin-1 and tumour necrosis factor: the role of Kupffer cells

Background/Aims: Injection into mice of a small dose of either a lipopolysaccharide or interleukin-1 induces a slowly developing accumulation of 5-hydroxytryptamine, predominantly in the liver. We have established that this 5-hydroxytryptamine accumulation is the result of the translocation of platelets to hepatic sinusoidal spaces and, further, into Disse spaces, and that the platelets make direct contact with hepatocytes. In the present study, we report our recent findings on this phenomenon.Methods: Platelets contain a large amount of 5-hydroxytryptamine, but the 5-hydroxytryptamine content of the liver is normally very small. Therefore, the translocation of platelets to the liver was assessed by measuring 5-hydroxytryptamine as in previous studies, and it was also analysed by electron microscopy.Results: Anti-platelets agents, such as heparin and inhibitors of prostaglandin synthesis, were ineffective in preventing the lipopolysaaccharide-induced accumulation of 5-hydroxytryptamine in the liver. Of the various cytokines tested, only interleukin-1 and tumour necrosis factor induced such an accumulation of 5-hydroxytryptamine. Intravenous injection of liposomes encapsulating dichloromethylene bisphosphonate resulted in an almost complete depletion of macrophages from the liver. The lipopolysaccharide- and cytokine-induced hepatic accumulations of 5-hydroxytryptamine were abolished almost completely in such macrophage-depleted mice. Electron microscopy revealed no accumulation of platelets in the liver after injection of lipopolysaccharide into the macrophage-depleted mice. Surprisingly, in normal mice injected with lipopolysaccharide, several platelets were found inside some hepatocytes, even though there was no visible damage to these hepatocytes. In fact, there were many polysomes around the degranulated platelets within the hepatocytes, suggesting an enhanced protein synthesis.Conclusion: These results suggest that, in response to lipopolysaccharide, interleukin-1 or tumour necrosis factor, platelets translocate into the liver in a way that is different from aggregation, and that some, at least, enter hepatocytes. During these processes, hepatic macrophages play an essential role.     

Evidence of direct interaction between Kupffer cells and colon cancer cells: an ultrastructural study of the co-culture

Abstract: A co-culture study of purified rat Kupffer cells and human colon cancer cells was performed, and the process of the tumor cell injury was observed under an inverted type fluorescence microscope loaded with propidium iodide, and also under an electron microscope. Ultrastructurally there was direct membrane-to-membrane interaction between Kupffer cells and colon cancer cells in time. The interaction occurred 1 h after start of the co-culture, and injured tumor cells were observed closely attached to pseudopodia of Kupffer cells at 6 h. The number of propidium iodide-positive tumor cells with damage increased in time. Pretreatment with N^G-monomethyl-L-arginine reduced the number of injured tumor cells without preventing morphological interactions, but superoxide dismutase did not prevent the tumoricidal effect. Pretreatment with trypsin completely inhibited cell interaction and damage to tumor cells. In conclusion, the morphological interaction of Kupffer cells as a first step and the involvement of nitric oxide-derived free radicals as a second step seem to play a significant role in the host-defense mechanism.     

Platelets attenuate production of cytokines and nitric oxide by macrophages in response to bacterial endotoxin

Considerable evidence has been accumulated concerning the roles of platelets in immune responses. In the present study, we examined the functional modulation of macrophages by platelets. When mouse bone marrow-derived macrophages (BMDMs) were co-cultured with platelets, BMDMs produced lower levels of nitric oxide (NO), tumor necrosis factor-α (TNF)-α, and interleukin (IL)-6 in response to a bacterial endotoxin (LPS) and zymosan. The attenuation in the macrophage susceptibility to LPS appeared to be mediated by soluble factors secreted from platelets. The mRNA levels of NOS2 (iNOS), TNF-α, and IL-6 in LPS-stimulated BMDMs that had been cultured with a conditioned medium of platelets were also decreased as analyzed by RT-qPCR. The ability of the platelet-conditioned medium to suppress macrophage NO production was recovered in a high-molecular-weight fraction (>670 kDa) after gel-filtration chromatography on a Superose 6 column. These results suggest that platelets control the susceptibility of macrophages to prevent excessive responses to LPS and provide mechanistic insight into a previous report that experimental thrombocytopenia aggravated organ failure in LPS-induced endotoxemia.     

Influence of Kupffer cells and platelets on ischemia-reperfusion injury in mild steatotic liver

AIM: To investigate the effect of mild steatotic liver on ischemia-reperfusion injury by focusing on Kupffer cells (KCs) and platelets. METHODS: Wistar rats were divided into a normal liver group (N group) and a mild steatotic liver group (S group) induced by feeding a choline-deficient diet for 2 wk. Both groups were subjected to 20 min of warm ischemia followed by 120 min of reperfusion. The number of labeled KCs and platelets in sinusoids and the blood perfusion in sinusoids were observed by intravital microscopy (IVM), which was performed at 30, 60 and 120 min after reperfusion. To evaluate serum alanine aminotransferase as a marker of liver deterioration, blood samples were taken at the same time as IVM.RESULTS: In the S group, the number of platelets adhering to KCs decreased significantly compared with the N group (120 after reperfusion; 2.9±1.1 cells/acinus vs 4.8±1.2 cells/acinus, P<0.01). The number of KCs in sinusoids was significantly less in the S group than in the N group throughout the observation periods (before ischemia, 19.6±3.3 cells/acinus vs 28.2±4.1 cells/acinus, P<0.01 and 120 min after reperfusion, 29.0±4.3 cells/acinus vs 40.2±3.3 cells/acinus, P<0.01). The blood perfusion of sinusoids 120 min after reperfusion was maintained in the S group more than in the N group. Furthermore, elevation of serum alanine aminotransferase was lower in the S group than in the N group 120 min after reperfusion (99.7±19.8 IU/L vs 166.3±61.1 IU/L, P=0.041), and histological impairment of hepatocyte structure was prevented in the S group. CONCLUSION: Ischemia-reperfusion injury in mild steatotic liver was attenuated compared with normal liver due to the decreased number of KCs and the reduction of the KC-platelet interaction.     

NRAMP1 and TNF-alpha polymorphisms and susceptibility to tuberculosis in Thais

OBJECTIVE AND BACKGROUND: Polymorphisms in the natural resistance-associated macrophage protein gene 1 (NRAMP1) and tumour necrosis factor (TNF)-alpha gene have been found to be associated with susceptibility to tuberculosis in different populations. However, the results are inconsistent. This study aimed to determine whether NRAMP1 and TNF-alpha variants are associated with tuberculosis in Thais. METHODS: Polymorphisms of NRAMP1 at INT4, D543N and the 3' untranslated region, and of TNF-alpha at +488, -238, and -308, were examined in 149 tuberculosis patients and 147 healthy controls. PCR using sequence-specific oligonucleotides and sequence-specific priming were used to genotype the NRAMP1 and TNF polymorphisms, respectively. RESULTS: There were no significant differences in the distribution of the genotype frequencies for the NRAMP1 and TNF-alpha polymorphisms between the patients and controls. CONCLUSIONS: The NRAMP1 and TNF-alpha genes are not associated with susceptibility to tuberculosis in Thais.     

沉默胆固醇调节原件结合蛋白2基因对炎症因子所致HepG2细胞内胆固醇异常积聚的影响

目的 探讨沉默胆固醇调节原件结合蛋白(SREBP)2对炎症因子所致HepG2细胞内脂质异常积聚的影响.方法 通过脂质体转染SREBP2-shRNA质粒、G418抗性筛选HepG2细胞,建立沉默SREBP2的HepG2稳定细胞株(SREBP2 shRNA-HepG2)及阴性对照质粒HepG2稳定细胞株(NC shRNA-HepG2).对两种细胞分别给予无血清培养处理(对照组)、炎症因子[肿瘤坏死因子(TNF)α20ng/ml]、高脂[低密度脂蛋白(LDL) 100 μ g/ml]和联合干预(20ng/ml TNFα +100μg/ml LDL)处理.油红O染色及酶法检测细胞中脂质积聚情况,实时定量PCR和Western blot 分别检测SREBP2及其下游靶基因低密度脂蛋白受体(LDLr)和3-羟-3-甲基戊二酰辅酶A(HMGCoA)还原酶的基因和蛋白表达情况.对数据用单因素方差分析及2×2×2析因设计方差分析比较. 结果 成功建立阴性对照稳定细胞株NC shRNA-HepG2及抑制SREBP2表达的稳定细胞株SREBP2shRNA-HepG2.在NC shRNA-HepG2细胞中,TNF α处理使细胞内胆固醇积聚增加,并能上调SREBP2下游靶基因LDLr、HMGCoA还原酶基因和蛋白的表达,其mRNA相对表达量分别为1.68±0.03、1.31±0.05,F值分别为107.42,59.08,P值均＜0.01 ;其蛋白相对表达量分别为1.49±0.10、1.54±0.06,F值分别为46.24,247.10,P值均＜0.01.而在SREBP2 shRNA-HepG2细胞中,TNFα对胞内胆固醇沉积的作用可以被明显减弱,进一步基因和蛋白检测结果显示,炎症因子TNF α对LDLr、HMGCoA还原酶的上调作用亦被明显抑制. 结论 炎症因子通过促进SREBP2及其下游靶基因表达致HepG2细胞内胆固醇异常积聚,而通过RNA干扰抑制SREBP2的表达,可以明显减轻炎症因子所致的细胞内胆固醇的异常积聚.     

Possible role of tumor necrosis factor-alpha in erythropoietic suppression by endotoxin and granulocyte/macrophage colony-stimulating factor

Injection of bacterial endotoxin or granulocyte/macrophage colony-stimulating factor (GM-CSF) into exhypoxic polycythemic mice simultaneously with erythropoietin (EPO) suppressed erythroid cell formation, as monitored by ⁵⁹Fe incorporation into circulating red blood cells. This effect was dose-dependent and time-dependent. GM-CSF did not inhibit erythroid cell formation directly, as the antibody to the GM-CSF did not neutralize the effect of endotoxin, the inducer of GM-CSF. The suppression of both agents could be partially corrected by prior injection of a monoclonal antibody to tumor necrosis factor α (anti-TNFα). These results indicate that the suppression of EPO-induced erythroid cell formation by endotoxin and GM-CSF was due in part to the production of TNFα. © 1996 Wiley-Liss, Inc.     

Induction of CINC (interleukin-8) production in rat liver by non-parenchymal cells

The production of interleukin-8 (CINC: cytokine-induced neutrophil chemo-attractant) from different cell populations in the rat liver was studied and cells related to the initiation of CINC production in lipopolysaccharide (LPS)-injected endotoxaemic rats were characterized. Sinusoidal endothelial cells (16.4 ± 10.6 ng/mL) produced significantly higher amounts of CINC in 24 h primary cultures compared with hepatocytes (0.9 ± 0.9 ng/mL; P < 0.05) and Kupffer cells (6.5 ± 5.1 ng/mL; P < 0.05). Lipopolysaccharide, tumour necrosis factor-α (TNF-α), and interleukin-1α (IL-1α) stimulated different liver cell populations to produce CINC; LPS mainly stimulated Kupffer cells, TNF-α stimulated hepatocytes and IL-1α stimulated all three types of cells. Intraperitoneal injection of LPS (4 mg/kg) caused CINC accumulation in non-parenchymal cells of the rat liver within 1 h of injection, as shown by immunohistochemical staining. In contrast, CINC-positive hepatocytes were not seen until 3 h after injection of LPS. Ethanol was not a direct inducer of CINC production by rat hepatocytes in vitro. These findings strongly suggest that non-parenchymal liver cells, including sinusoidal endothelial cells, are the main source of CINC. Our data also suggest that during endotoxaemia, CINC production is initiated by non-parenchymal cells and this is followed by production from hepatocytes.     

胆汁内、外引流术对梗阻性黄疸大鼠血清肿瘤坏死因子和肝脏库普弗细胞诱导型一氧化氮合酶表达的影响

目的 研究胆汁内、外引流术对梗阻性黄疸大鼠血清肿瘤坏死因子-α(TNF-α)水平和肝脏库普弗细胞诱导型一氧化氮合酶(iNOS)表达的影响.方法 采用成年雄性Sprague-Dawley大鼠48只,分成梗阻性黄疸、胆汁外引流、内引流和假手术四组,每组12只.采用原位灌注消化肝脏及贴壁培养方法 分离并纯化库普弗细胞,采用逆转录(RT)-PCR方法 检测库普弗细胞iNOS mRNA表达.用ELlSA方法 测定血清TNF-α含量.结果 梗阻性黄疸组血清TNF-α水平为(110.8±26.3)pg/ml,与假手术组的(88.4±17.9)pg/ml比较,差异有统计学意义(P=0.045).内引流组解除胆道梗阻后,血清TNF-α水平受到抑制,为(89.84±28.3)pg/ml,而胆汁外引流组却无此作用,为(118.6±22.7)Pg/ml,后者与梗阻性黄疸组比较差异无统计学意义(P=0.059).胆道梗阻形成后,梗阻性黄疸组库普弗细胞iNOSmRNA表达增强(0.82±0.24),显著高于假手术组(0.38±0.35,P=0.005).胆汁内引流术解除黄疸后,内引流术组的库普弗细胞iNOS mRNA表达受到抑制(0.59±0.35),但与梗阻性黄疸组比较差异无统计学意义(P=0.139),外引流组iNOSmRNA表达并未受抑制(0.974±0.48),与梗阻性黄疸组比较差异无统计学意义(P=0.321),但显著高于胆汁内引流组(P=0.016).结论 胆汁内引流术在逆转梗阻性黄疸大鼠升高的血清TNF-α水平和肝脏库普弗细胞iNOS mRNA的表达方面优于胆汁外引流术.