巨噬细胞移动抑制因子基因的克隆和原核表达

目的：扩增、克隆人巨噬细胞移动抑制因子（MIF）基因，并在大肠杆菌中表达出具有生物活性的重组MIF蛋白。方法：根据人MIF基因序列，设计、合成PCR引物，利用RT-PCR技术从人T淋巴细胞mRNA中扩增MIF基因。将MIF定向插入原核表达载体pGEX-4T-1，并将构建正确的重组表达载体pGEX-4T-MIF转化工程菌BL21(DE3)，用异丙基硫代-β-D-半乳糖（IPTG）诱导表达重组MIF蛋白。用GSTrap亲合柱纯化表达产物GST-MIF，行柱上凝血酶消化，洗脱获得MIF蛋白。用巨噬细胞移动抑制试验（MMI）鉴定MIF蛋白的生物活性。结果：限制性内切酶分析和DNA测序结果表明，成功构建了重组质粒pGEX-4T-MIF，人MIF cDNA长348 bp，编码115个氨基酸。经IPTG诱导，高效表达出可溶的GST-MIF蛋白。SDS-PAGE 和 Western blotting分析显示，GST-MIF经凝血酶消化，获得13 kU的MIF蛋白。MIF蛋白对巨噬细胞移动的抑制率达30%，具有生物活性。结论：克隆、测定了人MIF基因，在大肠杆菌表达出具有生物活性的MIF蛋白。     

Involvement of macrophage migration inhibitory factor gene in celiac disease susceptibility

The MIF gene has been associated with several diseases with inflammatory and autoimmune background, such as ulcerative colitis, rheumatoid arthritis and systemic lupus erythematosus. We aimed at testing the influence of two functional MIF promoter variants in celiac disease (CD) susceptibility. A (CAAT)(5-8) tetranucleotide repeat at position -794 and a single-nucleotide polymorphism at -173G/C were analyzed in the Spanish population (531 patients and 887 healthy controls). chi(2) statistics or Fisher exact test were used for comparisons. The -173C allele significantly increased risk ((CC+GC) vs GG: odds ratio (OR) (95% confidence interval (CI))=1.41 (1.10-1.81); P=0.005), as did carriage of the (CAAT)(7) allele (OR (95% CI)=1.36 (1.02-1.82); P=0.03) and of the haplotype (CAAT)(7)//-173C (OR (95% CI)=1.33 (1.00-1.76); P=0.04). Our data evidence for first time the role of the MIF gene increasing predisposition to CD. A common effect of MIF variants seems to underlie the etiology of these complex conditions.     

巨噬细胞移动抑制因子在瘢痕疙瘩中的表达

背景：巨噬细胞移动抑制因子参与了多种疾病的发生、发展过程,它在肿瘤、自身免疫性疾病、炎症反应、血管新生、纤维化等方面发挥重要作用,这些生物学特征与瘢痕疙瘩较为相似。目的：检测巨噬细胞移动抑制因子(MIF)在瘢痕疙瘩、增生性瘢痕、正常皮肤组织中的表达情况,说明巨噬细胞移动抑制因子分布位置和数量的差异。方法：收集临床病理性瘢痕切除后标本共40例,其中瘢痕疙瘩组20例,增生性瘢痕组20例,另取正常皮肤标本10例作为对照。分别做苏木精-伊红染色和免疫组织化学染色观察巨噬细胞移动抑制因子在病理性瘢痕及正常皮肤中的表达情况。结果与结论：巨噬细胞移动抑制因子在正常皮肤、增生性瘢痕、瘢痕疙瘩中均有表达,但在瘢痕疙瘩中表达明显强于增生性瘢痕和正常皮肤(P < 0.01)。说明巨噬细胞移动抑制因子在瘢痕疙瘩中的异常浸润可能与瘢痕疙瘩的形成有关。中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

巨噬细胞移动抑制因子的信号转导研究进展

巨噬细胞移动抑制因子(MIF)是一种重要的多功能细胞因子,在机体中分布广泛,可能参与了机体多种病理生理反应,其信号转导机制复杂而广泛,主要有激活细胞外信号调节激酶1/2(ERK1/2)信号转导途径、NF-κB途径,Jab 1途径等;与众多自身免疫疾病密切相关。     

Evidence of association of macrophage migration inhibitory factor gene polymorphisms with systemic lupus erythematosus

The aim of this study was to evaluate the potential association of functional polymorphisms of macrophage migration inhibitory factor with systemic lupus erythematosus. Our study includes 711 systemic lupus erythematosus (SLE) patients and 755 healthy controls. We genotyped the migration inhibitory factor (MIF) -173G/C using a polymerase chain reaction (PCR) system with predeveloped TaqMan allelic discrimination assay and the MIF -794 CATT(n) microsatellite polymorphism using a PCR-fluorescent method. A statistically significant difference in the distribution of the MIF -173(*)C allele between SLE patients and controls (P=0.004, OR=1.34, 95% CI=1.05-1.27) was observed. In addition, the frequency of the MIF -173(*)C/C genotype was higher in SLE patient (P=0.002, OR=2.58, 95% CI=1.32-5.10). No differences in the distribution of CATT(n) were found. However, the haplotypes analyses showed that only the CATT(7)-MIF -173(*)C haplotype was associated with a higher susceptibility to SLE (P=0.001, OR 1.84, 95% CI 1.35-2.79). No association with clinical features was detected in any case. These results suggest that both, MIF -173(*)C allele and CATT(7)-MIF -173(*)C haplotype, confer susceptibility to SLE in our population.     

Stimulation of vascular smooth muscle cell migration by macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) is a well known proinflammatory factor that influences the migration and proliferation of various cell types, predominantly monocytes and macrophages. Recent evidence suggests an important role for MIF in the progression of atherosclerosis and restenosis. For this reason, we studied the effect of MIF on platelet-derived growth factor-BB (PDGF-BB)-induced migration and PDGF receptor protein expression in vascular smooth muscle cells (VSMCs). Furthermore, the possibility of MIF influencing the migration of VSMCs was investigated. Our results show that short-term incubation of MIF is able to enhance PDGF-BB-induced migration. Long-term incubation decreases PDGF-BB-induced migration, but preserves a short-term stimulatory effect. These effects are not regulated at the level of PDGF receptor protein expression. MIF also acts as a chemoattractant for VSMCs, with a maximum response at 15 ng/ml. In contrast, the proliferation of VSMCs was unaffected by MIF. We conclude that MIF has a biphasic effect on VSMC migration. It remains unclear whether this effect is direct or involves the secretion of unidentified promigratory factors. Exogenous MIF does not stimulate VSMC proliferation; however, a role for MIF in proliferation cannot be fully ruled out. In view of the known key contributions of macrophage-derived MIF and VSMCs, the observed effects may well play a role in the progression of atherosclerosis and restenosis.     

Beta cell function: the role of macrophage migration inhibitory factor

During evolution, beta cells adapted to a sole aim: the production and stimulus-dependent secretion of insulin. This acquired specificity was accompanied by a loss of protection mechanisms predisposing beta cell to a high vulnerability. Among beta cell-damaging molecules, a new one has been identified recently: macrophage migration inhibitory factor (MIF). MIF was at first designated as a T-cell product that inhibits random movement of macrophages. Over the years, the number of functions attributed to this protein increased significantly, positioning MIF at the top of inflammatory cascade in the combat against infection and in immunoinflammatory and autoimmune diseases. This exceptionally versatile molecule regulates insulin secretion in physiological conditions, while in pathological states it alters beta cell function and induces their apoptosis or necrosis and affects beta cell neoplasia.     

Cycle-dependent expression of macrophage migration inhibitory factor in the human endometrium

BACKGROUND: Macrophage migration inhibitory factor (MIF) isa multifunctional cytokine that was shown to promote angiogenesisand tissue remodelling. Our previous studies identified MIFas one of the principal bioactive molecules involved in endothelialcell proliferation released by ectopic endometrial cells. METHODSAND RESULTS: In the present study, we examined the expressionof MIF in the human endometrium and found an interesting distributionand temporal pattern of expression throughout the menstrualcycle. Immunoreactive MIF was predominant in the glands andsurface epithelium. Dual immunofluorescence analysis furtheridentified endothelial cells, macrophages and T-lymphocytesas cells markedly expressing MIF in the stroma. Quantitativeassessment of MIF protein showed a regulated cycle phase-dependentexpression pattern. MIF expression increased in the late proliferative/earlySecretory phase of the menstrual cycle was moderate during thereceptive phase or what is commonly called the implantationwindow before increasing again at the end of the cycle. Thispattern paralleled MIF mRNA expression determined by northernblot. CONCLUSION: The cycle phase-specific expression of MIFsuggests a tight regulation and perhaps different roles forthis factor in the reparative, reproductive and inflammatory-likeprocesses that occur in human endometrium during every menstrualcycle.     

Purification of thymic macrophage migration inhibitory factor (MIF)

Aqueous extracts of the thymus of animals which had been challenged immunologically have been shown to contain MIF activity. This MIF could be purified by precipitation with 70% ethanol, concentrated by ultrafiltration between 30,000 and 50,000 daltons, isoelectrically focused at pH 6.8–7.1, and electrophoresed on preparative acrylamide gels. The resulting product is electrophoretically homogeneous at pH 4.3 in polyacrylamide-gel electrophoresis and SDS-gel electrophoresis. It has a molecular weight of 36,000 daltons. It is trypsin- and neuraminidase-labile and is thermostable. It degrades and reassembles in electrophoresis at pH 7.0. It is not chemotactic for macrophages but apparently activates them phagocytically. It has no proteolytic activity.Supported in part by a contract from the Office of Nival Research N00014-71-C-0203.     

Effects of antisense oligonucleotides on theexpression of macrophage migration inhibitoryfactor on macrophages

It is well known that the infiltration and prolifera tion of macrophages are the main features in manycases of experimental studies and human glomeru lonephritis, and they also play an important role inthe progressive renal injuries in the development ofglonerulonephritis [ 1 4 ]. Macrophage migrationfactor (MIF) is a proinflammatory cytokine thatwas originally described as a product of activatedlymphocytes that inhibited the random migration ofmacrophages in vitro and promoted the acc…     

Link between macrophage migration inhibitory factor and cellular redox regulation

Macrophage migration inhibitory factor (MIF) is an evolutionary conserved 12.5-kDa protein mediator with multiple functions in innate and acquired immunity. Upon leaderless secretion, MIF acts as a typical inflammatory cytokine, but there is no structural homology between MIF and any of the known cytokine protein families. Also, MIF is unique among cytokines in that it exhibits certain endocrine properties and has enzymatic activity. The catalytic thiol-protein oxidoreductase (TPOR) activity of MIF is mediated by a Cys-Ala-Leu-Cys active site between residues 57 and 60 that can undergo reversible intramolecular disulfide formation. Such a redox motif is typically found in TPORs of the thioredoxin (Trx) family of proteins. MIF seems to act as a disulfide reductase, and structure-function analyses of the redox site indicate that this activity is not only observed in vitro, but plays a role in cellular redox homeostasis, apoptosis inhibition, MIF-mediated monocyte/macrophage activation, and possibly the modulation of the activity of MIF-binding proteins. In this Forum review, the biochemical and biological evidence for a role of the TPOR activity for various MIF functions is summarized and discussed. In particular, the marked functional homologies with Trx proteins, the MIF redox/MHC II link, and recent attempts to discern the intra- versus extracellular roles of the MIF TPOR activity are dealt with.     

巨噬细胞移动抑制因子与疾病相关性研究进展

巨噬细胞移动抑制因子(MIF)主要由巨噬细胞产生,T淋巴细胞、单核细胞、树突状细胞、B淋巴细胞、中性粒细胞、嗜酸性细胞、嗜碱性细胞都可以表达MIF.MIF的主要生物效应是抑制巨噬细胞的游走,促进巨噬细胞在炎症局部的聚集、浸润、增生、活化及分泌一些炎性细胞因子如NO的释放,COX-2的诱导,在发挥免疫功能的同时加重炎症损伤.因而MIF在巨噬细胞趋化和致炎过程中发挥着举足轻重的作用,作为疾病生物标志物和治疗靶标具有重要意义.     

Participation of immunoglobulin-bearing lymphocytes in the production of macrophage migration inhibitory factor

The role of immunoglobulin-bearing cells in the production of macrophage migration inhibitory factor (MIF) by tuberculin-stimulated lymphocytes of guinea pigs, immunized with complete Freund's adjuvant, was studied. It was found that: (1) pretreatment of lymphocytes with rabbit anti-guinea pig IgG (anti-IgG) does not block antigen-induced MIF production. (2) Passage of lymphocytes through double layer IgG-anti-IgG gelatin bead columns (the preparation of which is described) abolishes MIF formation by the eluted cells. Cells retained on the columns can be recovered and where shown to produce MIF, when stimulated by antigen. (3) Pulsing of lymphocytes with anti-IgG, for 2 h at 37°C, results in MIF synthesis by the cells cultured in medium, in the absence of specific antigen. These findings indicate that cells bearing Ig or Ig fragments are either able to secrete MIF themselves, upon stimulation with antigen or anti-IgG, or are required for MIF production by a different cell type.     

巨噬细胞移动抑制因子-173G/C多态性与冠心病的相关性研究

目的研究巨噬细胞移动抑制因子（macrophage migration inhibitory factor, MIF）基因MIF 5′非翻译区-173G／C多态性在中国南方汉族人群中的分布及与冠心病的相关性。方法采用聚合酶链反应-限制性片段长度多态性技术，对汉族138例冠心病患者及163名正常人群MIF基因-173G／C位点进行研究，对于限制酶酶切结果再进行DNA测序鉴定。结果冠心病患者与正常对照中只检出-173GG和-173GC基因型，均未检出-173CC基因型。正常人群和冠心病患者的MIF-173G等位基因频率分别为0．966和0．917，MIF-173C等位基因频率分别为0．034和0．083，冠心病患者MIF-173GC基因型频率（0．167）明显高于对照组（0．068）（OR：2．764，95％CI：1．295～5．899；P=0．007）。结论MIF基因-173G／C位点多态性与冠心病有关，C等位基因可能是汉族人群冠心病的易感性标志。     

氧化型低密度脂蛋白诱导巨噬细胞移动抑制因子的表达

目的:探讨氧化型低密度脂蛋白(ox-LDL)对人巨噬细胞分泌巨噬细胞移动抑制因子(MIF)的影响。方法:体外培养巨噬细胞株28SC,在其培养基中加入终浓度为150mg/L的氧化型低密度脂蛋白进行时间效应实验,37℃共育后,分别于0h、3h、6h、12h、24h、36h收集细胞和培养介质;在其培养基中分别加入终浓度为0mg/L、15mg/L、30mg/L、75mg/L、150mg/L、300mg/L的氧化型低密度脂蛋白进行剂量效应实验,37℃共育24h后收集细胞和培养介质。采用RT-PCR和ELISA分别测定MIFmRNA和蛋白表达水平。结果:氧化型低密度脂蛋白可诱导培养巨噬细胞分泌MIFmRNA和蛋白,并呈时间和剂量依赖性增加。结论:MIF可能参与氧化型低密度脂蛋白所致的动脉粥样硬化进程。     

血管紧张素转换酶2基因转染对人内皮细胞MIF表达的影响

目的：探讨重组血管紧张素转换酶2（ACE2）基因转染对体外培养的人血管内皮细胞中由血管紧张素（Ang）II诱导的巨噬细胞移动抑制因子（MIF）表达的影响。方法:克隆和构建含人ACE2基因全长的重组质粒（pACE2），并将之转染入人血管内皮细胞中。分别采用实时定量PCR和Western印迹技术检测转染细胞中的MIF mRNA与蛋白表达情况。结果: Ang Ⅱ（100 nmol/L）和Ang IV（100 nmol/L）刺激后均可诱导人血管内皮细胞中MIF mRNA及蛋白表达增加（P<0.01）。pACE2基因转染可明显抑制内皮细胞中由Ang II和Ang IV诱导的MIF mRNA和蛋白表达（P<0.05）。结论: ACE2基因过表达可明显抑制人内皮细胞中炎症介质MIF的表达，提示ACE2基因具有一定的抗炎症效应。通过调节ACE2基因的活性和表达，很可能为炎症相关疾病如动脉粥样硬化治疗提供新的策略。     

The effect of helicobacter pylori eradication on macrophage migration inhibitory factor, c-reactive protein and fetuin-a levels

OBJECTIVES:

To determine the effect of Helicobacter pylori (H. pylori) eradication on blood levels of high-sensitivity C-reactive protein (hs-CRP), macrophage migration inhibitory factor and fetuin-A in patients with dyspepsia who are concurrently infected with H. pylori.

METHODS:

H.pylori infection was diagnosed based on the ¹⁴C urea breath test (UBT) and histology. Lansoprazole 30 mg twice daily, amoxicillin 1 g twice daily, and clarithromycin 500 mg twice daily were given to all infected patients for 14 days; ¹⁴C UBT was then re-measured. In 30 subjects, migration inhibitory factor, fetuin-A and hs-CRP levels were examined before and after the eradication of H. pylori infection and compared to levels in 30 healthy subjects who tested negative for H. pylori infection.

RESULTS:

Age and sex distribution were comparable between patients and controls. Migration inhibitory factor and hs-CRP levels were higher, and fetuin-A levels were lower, in H. pylori-infected patients (p<0.05). Following eradication of H. pylori, migration inhibitory factor and hs-CRP levels were significantly decreased, whereas fetuin-A levels were increased. However, eradication of the organism did not change lipid levels (p>0.05).

CONCLUSION:

These findings suggest that H. pylori eradication reduces the levels of pro-inflammatory cytokines such as migration inhibitory factor and hs-CRP and also results in a significant increase in anti-inflammatory markers such as fetuin-A.     

巨噬细胞移动抑制因子对固有免疫的调节作用

长期以来,巨噬细胞移动抑制因子(MIF)一直是一种功能不明的细胞因子.最近几年的研究发现,MIF是宿主抗微生物预警系统和应急反应(可以促进免疫细胞促炎症功能)的一个必要组成成份,是一种对固有免疫反应具有关键性调节作用的重要分子.研究表明,MIF参与了包括脓毒症、炎症和自身免疫性炎症疾病在内的一系列疾病的发病机制,这为未来使用靶向MIF疗法治疗相关的人类疾病提供了全新的思路.