STAT3在肝细胞增殖过程中对C/ebpβ表达的影响

目的探讨STAT3在IL-6刺激肝细胞增殖过程中对C/ebpβ表达变化的影响。方法用含15 ng/ml的IL-6培养基培养大鼠正常肝细胞BRL-3A,运用CCK试剂盒检测该细胞在IL-6刺激后不同时间里的增殖情况,Western blot技术检测STAT3、P-STAT3、C/ebpβ的蛋白表达变化情况;利用siRNA瞬时转染技术抑制STAT3在BRL-3A细胞中的表达并观察IL-6刺激肝细胞增殖过程中C/ebpβ的表达变化。结果 IL-6能有效的刺激肝细胞增殖,在增殖早期STAT3、P-STAT3、C/ebpβ表达均明显上调;在肝细胞增殖过程中沉默STAT3后C/ebpβ表达下调。结论在IL-6肝细胞增殖过程中STAT3对C/ebpβ起正向调控作用。     

IL-10对大鼠原代肝细胞增殖的影响

目的:探讨IL-10对体外培养大鼠原代肝细胞增殖的影响。方法:胶原蛋白酶原位灌流法分离大鼠肝细胞,RT-PCR法检测肝内不同细胞标志基因的表达情况,对比原代肝细胞与大鼠正常肝组织的检测结果,进行细胞纯度鉴定;RT-PCR法检测原代肝细胞IL-10和IL-10受体α(IL10Rα)的表达情况;原代肝细胞分3组培养:正常组(N组)、对照组(C组)和IL-10干预组(I组);通过MTT分析、台盼兰细胞计数以及流式细胞术检测DNA含量等方法,了解外源性IL-10对肝细胞增殖的影响。结果:细胞鉴定结果显示,所有的标志基因在肝组织都可检测到有较高的表达,原代肝细胞虽高表达肝细胞标志基因,而肝内其他细胞的标志基因则不表达或低表达。RT-PCR检测显示原代肝细胞可表达IL-10和IL10Rα。台盼兰细胞计数提示IL-10干预48h,I组平均细胞数仅为C组的71.96%(P<0.05);MTT检测亦显示IL-10干预24、48h,I组吸光度值分别为C组的88.41%与90.24%(P<0.05);流式细胞术检测结果表明IL-10干预24h,I组肝细胞峰DNA含量是C组的59.06%,I组较C组降低(P<0.01),甚至低于N组(P<0.01)。结论:分离的原代肝细胞纯度较高,大鼠原代肝细胞表达IL-10/IL10R mRNA,IL-10抑制大鼠原代肝细胞增殖。     

组织蛋白酶B在大鼠肝脏的分布

目的研究组织蛋白酶B(cathepsins B,CB)在正常SD大鼠肝脏组织中的表达。方法采用免疫荧光组织化学的方法,观察CB在大鼠肝脏组织中的分布。结果 CB免疫反应阳性细胞主要分布在肝细胞、枯否细胞、肝窦内皮细胞,枯否细胞、肝窦内皮细胞的免疫活性比较强,肝细胞的免疫活性比较弱,而肝星状细胞没有CB免疫活性。阳性物质分布于细胞质,细胞核阴性。结论 CB在大鼠肝脏组织的表达提示其可能在肝脏起着非常重要的作用。     

大鼠肝癌发生过程中转化生长因子—α在肝组织中的表达

以二乙基亚硝胺诱发大鼠肝癌、用免疫组织化学法检测肝组织中转化生长因子－α的表达变化。结果显示：正常大鼠肝的枯否细胞呈转化生长因子－α阳性表达；至诱癌第８周，除少量枯否细胞仍呈转化生长因子－α阳性外，部分肝细胞亦呈转化生长因子－α阳性，且吾两种反应类型，即部分肝细胞质和胞膜均呈转化生长因子－α阳性。有的肝细胞仅胞膜呈转化生长－α阳性。随着肝癌病变发展，转化生长因子－α阳必肝细胞数渐增多，转化生长因子     

肝细胞生长因子在肿瘤坏死因子相关凋亡诱导配体作用下对原代肝星状细胞增殖及凋亡的影响**☆

背景：活化的肝星状细胞是肝纤维化的关键因素,研究表明肝细胞生长因子能促进星状细胞凋亡,其具体机制可能与增强肿瘤坏死因子相关凋亡诱导配体(TRAIL)诱导星状细胞凋亡有关。 目的：观察肿瘤坏死因子相关凋亡诱导配体作用下,肝细胞生长因子对原代肝星状细胞增殖、凋亡的影响并初步探讨其可能机制。 方法：将SD大鼠原代肝星状细胞复苏、传代,细胞增殖明显时用于实验。实验分为4组：空白对照组为单纯肝星状细胞培养;肝细胞生长因子组：将100 μg/L肝细胞生长因子作用于肝星状细胞;TRAIL组：将2 mg/L的TRAIL作用于肝星状细胞;肝细胞生长因子+TRAIL组：将肝细胞生长因子预先刺激肝星状细胞24 h,再加入2 mg/L TRAIL。 结果与结论：MTT检测显示肝细胞生长因子及TRAIL分别在50~200 μg/L、0.5~1.5 mg/L各浓度下对肝星状细胞增殖抑制率无影响,TRAIL在2 mg/L作用下对肝星状细胞有抑制作用。流式细胞仪检测肝细胞生长因子+TRAIL组的中晚期凋亡率明显高于空白对照组及肝细胞生长因子组(P < 0.05);肝细胞生长因子+TRAIL组DR5荧光强度明显高于其他3组(P < 0.01)。提示在TRAIL作用下,肝细胞生长因子能促进肝星状细胞的凋亡、抑制其增殖。可能与肝细胞生长因子上调活化肝星状细胞表面DR5表达有关。      

重组肝刺激因子对肝脏卵圆细胞增殖的作用

目的观察重组人肝刺激因子(hHSS)对肝脏卵圆细胞增殖的影响。方法分离大鼠肝卵圆细胞，加以培养，并进行细胞学鉴定。将重组的hHSS作用于肝脏卵圆细胞，以MTT法、流式细胞术观察其对卵圆细胞的增殖作用。结果实验分离的卵圆细胞同时表达肝细胞和胆管细胞特异性标志物。给予重组hHSS作用12～24h后，细胞增殖速度减缓，细胞DNA合成减弱，其作用的最有效浓度为240mg/L。结论重组hHSS能抑制卵圆细胞的增殖。     

大鼠再生肝提取物对肝再生中肝细胞增殖的影响

大鼠肝大部(67%)切除24小时后取残肝匀浆,经加热,酒精沉淀、透析等步骤制备提取物,腹腔注射给正常大鼠和32%肝切术后的大鼠。光镜下观察肝组织切片,计算肝细胞有丝分裂百分率。鼠再生肝提取物可使32%肝切术后24-48小时再生肝细胞有丝分裂百分率明显增加,尤其在术后30小时可达对照组的3.9倍。结果表明,再生肝提取物中含有能够促进肝细胞增生的肝再生刺激因子(HSS),此因子对增殖前期(G_0/G_1早期)细胞作用不明显。     

DC固有胆碱能系统与JIA炎症免疫反应的关系

目的 探讨树突状细胞(DC)固有胆碱能系统与幼年特发性关节炎(JIA)炎症免疫反应的关系.方法 分离正常小鼠骨髓细胞,体外采用细胞因子诱导、分化,并刺激其成熟.通过细胞形态变化和表面分子对细胞鉴定;流式细胞术检测正常血清、JLA活动期血清和JIA活动期血清+美加明(MEC)3组血清对DC nAChRα7表达的影响;ELISA方法检测三组血清作用DC 18 h前后,培养上清中IL-12含量;MTT法和流式细胞术分别检测正常血清刺激的DC条件培养液组、JIA活动期血清刺激的DC条件培养液组、MEC干预的JIA活动期血清刺激的DC条件培养液组对小鼠脾淋巴细胞增殖活化的影响.结果 3组血清作用于成熟DC 18 h后DC nAChRα7表达为,JIA活动期血清组比正常血清组明显增强(P<0.05),而MEC作用后,nAChRα7表达有下调趋势;三组血清作用于成熟DC 18 h前后,培养上清中IL-12含量分别为,JIA活动期血清组比正常血清组IL-12含量均明显增高(P<0.01),而培养后,MEC干预组IL-12含量较JIA活动期血清组更高(P<0.05);3组血清刺激的DC条件培养液联合小剂量ConA对小鼠脾淋巴细胞增殖结果(SI值)和CD69表达是,MEC干预组增殖作用和CD69表达最强,JIA活动期血清刺激的DC条件培养液组次之.与正常血清刺激的DC条件培养液组比较增殖作用显著(P<0.01).结论 JIA活动期血清能刺激DC,使nAChRα7表达上调;MEC则下调DC nAChRα7表达,同时,伴有IL-12等炎性因子分泌及淋巴细胞增殖活化增强.故JIA的发生和活化可能与抑制DC胆碱能系统的抗炎作用有关,恢复胆碱能系统的正常调节可能是JIA治疗方向之一.     

心房肽对自发性高血压大鼠免疫细胞增殖活性的影响

本文探讨了心房肽对免疫细胞增殖活性的影响。结果表明,心房肽可以直接促进大鼠胸腺细胞的增殖反应,亦可协同ConA促进脾细胞及T细胞的增殖反应,协同IL-2促进经ConA诱导的淋巴母细胞增殖;心房肽还可促进ConA诱导的大鼠脾细胞产生IL-2。但心房肽并不影响B细胞的增殖反应,也不影响巨噬细胞产生IL-1。同时发现,自发性高血压大鼠脾细胞和胸腺细胞对心房肽与ConA协同刺激的增殖反应性低下。     

肝纤维化时肝脏OPN和PAI-1的表达变化

目的:研究骨桥蛋白(OPN)及纤溶酶原激活物抑制物(PAI-1)的表达特征及其在肝纤维化时的变化。方法:采用二甲基亚硝胺制作大鼠肝纤维化模型,大鼠肝脏常规HE和天狼猩红染色,采用SABC法做免疫组织化学染色及Western blotting检测OPN和PAI-1蛋白表达,抽提肝组织总RNA,RT-PCR检测OPN mRNA表达。结果:正常大鼠肝组织OPN和PAI-1表达极弱,肝纤维化大鼠肝脏中OPN表达增强,阳性信号散在或弥漫性分布,主要见于小叶内中央静脉周围、纤维间隔内以及周围巨噬细胞胞浆、枯否氏细胞、汇管区的部分肝细胞、肝窦壁内皮细胞。PAI-1在肝纤维化大鼠肝组织汇管区、肝细胞变性坏死处,肝窦周Disse间隙及毗邻以上部位的肝细胞,组织纤维间隔处及其外周细胞亦见阳性染色。Western blotting检测正常大鼠肝脏OPN的蛋白表达极低,肝纤维化组OPN的蛋白表达较正常组显著增强(P0.01)。与正常组比,肝纤维化组PAI-1表达也显著增强。RT-PCR检测结果显示,正常大鼠肝脏OPN mRNA表达极低,肝纤维化大鼠肝脏OPN mRNA的表达明显增强(P0.05)。研究结果证明,肝纤维化时大鼠肝组织OPN及PAI-1的表达水平显著增高。结论:肝纤维化时大鼠肝组织OPN及PAI-1的表达水平显著增高,OPN可能会促进PAI-1的高表达,从而抑制ECM降解、加速肝纤维化进程。     

Effect and mechanisms of FR167653, a dual inhibitor of TNF-α and IL-1, on BCG plus LPS induced-liver injury

Objective: To investigate the effect of FR167653, a dual inhibitor of tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1), on Bacillus Calmétte-Guérin (BCG) plus lipopolysaccharide (LPS) induced-liver injury and its mechanisms. Material and methods: Mouse liver injury was established by tail vein injection of 2.5 mg BCG, and 10 d later with 10 μg LPS. The alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were assayed by spectrophotometry. Liver samples were stained with hematoxylin and eosin. Rat hepatocytes (HCs) and Kupffer cells (KCs) were isolated by collagenase IV and pronase perfusion, and purified by density gradient separation. TNF-α and IL-1 concentrations were measured with ELISA. TNF-α and IL-1 mRNA in KCs was analyzed with RT-PCR. Results: FR167653 significantly decreased the elevated transaminase (ALT, AST) activity in serum of liver injured mice. Meanwhile, the degree of inflammatory cell infiltration and liver cell necrosis was also ameliorated. TNF-α and IL-1 production by KCs stimulated with LPS was significantly inhibited by FR167653. RT-PCR analysis demonstrated that FR167653 also reduced the augmented expression of TNF-α and IL-1 mRNA in KCs. However, FR167653 up to 10 μmol/L did not have a toxic effect on KC viability. In addition, FR167653 alleviated the HC injury induced by LPS pre-treated Kupffer cell-conditioned medium (KCCM). Addition of anti-IL-1 and anti-TNF-α MAbs significantly decreased the ALT level released from HCs incubated with LPS or FR167653 pre-treated KCCM. Conclusions: TNF-α and IL-1 released from activated KCs were involved in BCG plus LPS induced liver injury. FR167653 significantly attenuated hepatocyte injury via inhibition of TNF-α and IL-1 released from activated KCs. Hong Wei YAO and Li YUE contributed equally to this work. Received 19 May 2005; returned for revision 11 July 2005; accepted by I. Ahnfelt-R?nne 17 August 2005     

The stimulation of regenerative processes in the liver in acute liver failure

The efficiency of the newborn allogenic hepatocytes (NAH) in the treatment of the acute liver failure (ALF) in dogs is studied histologically, histochemically and ultrastructurally. The administration of NAH prevents, in part, the ischemic cell death and is efficient in the stimulation of the liver regeneration in ALF. NAH enhance hepatocyte and Kupffer cell proliferation, reduce the number of degenerative and necrotic foci, increase the number of cell organelles, their volume, compensate the function of the damaged liver, facilitate the morpho-functional restoration of the recipient ischemically damaged liver.     

Heparanase enhances early hepatocyte inclusion in the recipient liver after transplantation in partially hepatectomized rats

Hepatocyte transplantation is an emerging approach for the treatment of liver diseases. However, broad clinical application of this method has been limited by restricted source of cells and low efficiency of cell integration within the recipient liver. Heparanase cleaves heparan sulfate proteoglycans in the extracellular matrix and basement membrane, activity that affects cellular invasion associated with cancer metastasis and inflammation. This activity has a multifunctional effect on cell-cell interaction, cell adhesion, and angiogenesis. All these factors are important for successful integration of transplanted hepatocytes. Male donor hepatocytes pretreated with heparanase or untreated were transplanted into recipient female rat spleen following partial hepatectomy. Engraftment efficacy was evaluated by PCR for Y chromosome, histology and PCNA, and heparanase immunohistochemistry. In addition, proliferative activity of hepatocytes in vitro was determined by bromodeoxyuridine immunostaining. The number of heparanase-treated cells detected in the recipient liver was significantly increased three- to fivefold within 24-48 h posttransplantation and twofold at 14 days compared with untreated cells. The transplanted hepatocytes treated with heparanase were clearly seen inside portal vein radicles as cell aggregates up to 72 h posttransplantation. The number of portal radicles filled with heparanase-treated hepatocytes was increased compared to control early after transplantation. Heparanase treatment enhanced hepatocyte and sinusoidal endothelial cell proliferation in the liver, and hepatocyte proliferation within the spleen tissue. Preliminary in vitro studies with isolated hepatocytes treated with heparanase showed increased proliferative activity within 24-48 h of cell culture. These results suggest that preincubation of hepatocytes with heparanase increases the presence of hepatocytes within the recipient liver early following cell transplantation and stimulates both hepatocyte and sinusoidal endothelial cell proliferation.     

Hepatocyte modulation of Kupffer cell prostaglandin E2 production in vitro

It is likely that dynamic interactions between hepatocytes and Kupffer cells contribute to the responses of these cell types both under normal conditions and during sepsis. In this study, we examined the influences of hepatocytes on the concentration of the inflammatory mediator PGE2 in Kupffer cell cultures. Evidence to suggest that cultured rat hepatocytes both metabolize PGE2 and produce a substance that promotes LPS-stimulated Kupffer cell PGE2 biosynthesis include the following: 1) PGE2 levels in Kupffer cell: hepatocyte coculture were lower than the levels in Kupffer cell cultures early after LPS stimulation; 2) 36 h after LPS, coculture PGE2 levels exceeded the levels in Kupffer cell cultures despite the demonstrated capacity for hepatocytes to metabolize PGE2; 3) a transferable, non-dialyzable, and heat-unstable factor in hepatocyte supernatant promoted PGE2 production when added to Kupffer cells with LPS or after LPS; 4) there was no increased PGE2 synthesis when the hepatocyte supernatant was added without LPS or if hepatocyte supernatant was preincubated with the Kupffer cells for 6 or 18 h before LPS administration; 5) there was an inability of the hepatocyte factor to promote PGE2 production in response to other macrophage-activating agents, including calcium ionophore A23187 or phorphol myristate acetate; and 6) there was no increased cell replication or protein synthesis in the Kupffer cell cultures following hepatocyte supernatant incubation. The increased Kupffer cell PGE2 production by the hepatocyte supernatant was not due to contamination of the hepatocyte supernatant by endotoxin or PGE2. These in vitro results raise the possibility that hepatocytes have the capacity to modulate local PGE2 levels by two distinct mechanisms. Hepatocytes can metabolize PGE2 as well as release factor(s) which promote LPS-induced PGE2 production by Kupffer cells.     

Effects of acute moderate exercise on the phagocytosis of Kupffer cells in rats

AIM: This study investigated the function of Kupffer cells, and particularly their role as immunocompetent cells that come into contact with gut-derived endotoxin, in the acute exercise of rats. METHODS: Female Fischer 344 rats were run on a treadmill at 21 m min(-1) for 60 min on a 15% grade. RESULTS: Afterwards, the number of latex particles in the liver was higher in the exercising rats than that in resting rats and an increase in the number of latex particles phagocytosed by each Kupffer cell was noted. The plasma endotoxin concentration was significantly higher in the exercise group than in the resting rats, and the small intestine was damaged by the exercise. Plasma corticosterone and thyroxine 4 levels were unchanged. Although the number of Kupffer cells was unchanged by acute exercise, the number of CD14-positive Kupffer cells increased. Plasma liver enzyme activities were slightly increased by acute exercise, whereas plasma tumour necrosis factor-alpha was not detected. CONCLUSION: These results suggest that moderately intense exercise increases the phagocytosis of Kupffer cells, and that it might be induced by endotoxemia of portal blood caused by intestinal mucosal lesions resulting from acute exercise.     

High incidence of autoimmune dacryoadenitis in male non-obese diabetic (NOD) mice depending on sex steroid

Sinusoidal endothelial cells and Kupffer cells are the first cell populations in the liver that come into contact with gut-derived endotoxin in portal blood. Although endotoxin concentrations as high as 1 ng/ml are physiologically present in portal blood, no local inflammation is seen. We show that the proinflammatory cytokine IL-6, which is central to the development of inflammatory reactions in the liver, is produced by sinusoidal endothelial cells and Kupffer cells in response to low concentrations of endotoxin (100 pg/ml to 1 ng/ml). The anti-inflammatory cytokine IL-10 down-regulated endotoxin-induced IL-6 release in endothelial and Kupffer cells. Importantly, Kupffer cells secreted IL-10 after endotoxin stimulation and may therefore participate in the local regulation of inflammation. We have found that IL-6 secretion in Kupffer cells is tightly regulated by endogenous IL-10, because increased IL-6 secretion resulted when neutralizing antibodies to IL-10 were added to resting and endotoxin-challenged Kupffer cells. Furthermore, repeated exposure of endothelial cells to endotoxin induced a state of tolerance which resulted in decreased release of IL-6 in response to a second endotoxin challenge. Our results support the notion that inflammatory reactions in the liver in response to endotoxin are down-regulated by local release of the anti-inflammatory cytokine IL-10 that is produced by Kupffer cells.     

Kupffer cell proliferation and glucan-induced granuloma formation in mice depleted of blood monocytes by strontium-89

In mice with prolonged severe monocytopenia induced by selective irradiation of the bone marrow with the bone-seeking isotope 89Sr, the proliferative capacity of Kupffer cells was studied by immunohistochemistry with an anti-mouse macrophage monoclonal antibody, F4/80, ultrastructural peroxidase (PO) cytochemistry, and tritiated thymidine (3HTdR) autoradiography. The number and 3HTdR uptake of Kupffer cells were significantly increased in the splenectomized mice after severe monocytopenia had continued for more than 4 wk, and almost all the Kupffer cells showed a localization pattern of PO activity similar to that of resident macrophages in the liver of normal mice. In the glucan-induced granuloma formation in similar monocytopenic mice, Kupffer cells proliferated, conglomerated, and transformed into epithelioid cells, which fused together to become multinuclear giant cells. These results suggest that Kupffer cells are a self-renewing population by their own cell division and can participate actively in granulomatous inflammations in severely monocytopenic and intact mice.     

Activation of protease-activated receptor 2 stimulates proliferation and interleukin (IL)-6 and IL-8 secretion of endometriotic stromal cells

BACKGROUND: Inflammation has been proposed to play essential roles in the pathophysiology of endometriosis, in which neutrophils and mast cells have been suggested to be involved. We studied whether the protease-activated receptor 2 (PAR2), which is activated by enzymes from neutrophils and mast cells, in endometriotic stromal cells (ESC) has any implication in the development of the disease. METHODS: Cultured ESC were stimulated with various concentrations of a specific PAR2 agonist peptide. Proliferating activity of the cells was determined using immunostaining of proliferating cell nuclear antigen (a cell proliferation marker), 5-bromo-2'-deoxyuridine incorporation into DNA and cell count. The concentrations of interleukin (IL)-6 and IL-8 were measured using specific enzyme-linked immunosorbent assay kits. The phosphorylation of three mitogen-activated protein kinases (MAPK), i.e. p38 MAPK, p42/44 MAPK and stress-activated protein Kinase/c-jun N terminal Kinase, in ESC was examined with Western blot analysis. RESULTS: Activation of PAR2 stimulated the proliferation of ESC and the secretion of IL-6 and IL-8 from ESC in a dose-dependent manner. Activation of PAR2 stimulated the phosphorylation of all three MAPK, and inhibitors of each MAPK suppressed the PAR2 activation-induced proliferation of ESC. CONCLUSIONS: The activation of PAR2 in ESC may be involved in the pathophysiology of endometriosis by inducing the growth and inflammation of endometriotic lesions.