表没食子儿茶素-3-没食子酸酯抑制脂多糖诱导的巨噬细胞促炎因子TNF-α和IL-1β基因表达

表没食子儿茶素-3-没食子酸酯(epigallocatechin-3-gallate,EGCG)具有抗氧化、抗癌、抗炎等多种生物学特性,但对巨噬细胞中表达TNF-α及IL-1β的报告尚存在争议.本文旨在探索EGCG对脂多糖(LPS)诱导的小鼠腹腔巨噬细胞和RAW264.7细胞促炎细胞因子Tnf-α和Il-1β基因表达的影响.MTT结果显示,0~100μmol/L EGCG对RAW264.7细胞活力没有影响;实时荧光定量PCR(qRT-PCR)和ELISA分析显示,1 mg/L LPS可显著升高小鼠腹腔巨噬细胞和RAW264.7细胞Tnf-α和Il-1βmRNA和蛋白水平,EGCG单独处理对巨噬细胞Tnf-α和Il-1β的基因表达与蛋白生成没有影响,但可以抑制LPS诱导的巨噬细胞Tnf-α和Il-1β的基因表达与蛋白生成,并存在剂量依赖效应.上述结果提示,EGCG可以剂量依赖方式抑制LPS诱导的巨噬细胞促炎细胞因子Tnf-α和Il-1β的表达,这可能与EGCG的抗炎效应有关.     

EV-A71感染小鼠巨噬细胞诱导促炎细胞因子和趋化因子反应

为初步探索EV-A71在小鼠巨噬细胞中的复制情况和抗病毒的固有免疫应答,本文以小鼠巨噬细胞系RAW264.7为细胞模型,通过建立EV-A71绝对定量qPCR方法检测EV-A71病毒载量;EV-A71和紫外灭活的EV-A71感染RAW264.7,不同时间点提取总RNA,RT-qPCR检测促炎细胞因子、趋化因子和模式识别受体的mRNA表达变化水平。本研究成功建立了EV-A71的绝对定量qPCR检测方法,并发现EV-A71感染RAW264.7后随着感染时间的延长,EV-A71病毒载量呈递减趋势;EV-A71和紫外灭活的EV-A71可以引起IL-1β、IL-6、TNF-α促炎细胞因子和IP-10、MCP-1、MIP-1α趋化因子反应,上调TLR2、TLR1、TLR6、MDA5和RIG-I mRNA表达。研究结果显示,EV-A71在小鼠巨噬细胞中具有较低水平的复制,同时产生促炎细胞因子和趋化因子反应。     

黄芪甲苷Ⅳ对内毒素诱导RAW264.7细胞损伤的保护作用及机制研究

目的:研究黄芪皂苷Ⅳ对LPS诱导的巨噬细胞RAW264.7损伤的保护作用及机制.方法:测定细胞活力判断心肌细胞损伤的程度.TNF-α,IL-1β,IL-6,IL-10的释放以及NF-кB蛋白、Akt和磷酸化Akt表达用以研究作用机制.结果:黄芪皂苷Ⅳ对LPS引起的RAW264.7细胞损伤具有显著的抑制作用,1,3和10μM黄芪皂苷Ⅳ可显著降低LPS诱导的RAW264.7细胞TNF-α,IL-1β和IL-6的生成,促进IL-10的释放.黄芪皂苷Ⅳ剂量依赖性地增加了由LPS刺激而引起的RAW264.7细胞NF-kB蛋白的表达,抑制了LPS所致的p-Akt蛋白表达的升高.结论:黄芪皂苷Ⅳ可通过Akt-NF-kB途径调控促炎因子和抗炎因子表达的失衡,有效发挥对LPS诱导巨噬细胞RAW264.7损伤的保护作用.     

甘草酸对巨噬细胞抗绵羊肺炎支原体感染的调控作用研究

为观察甘草酸在小鼠巨噬细胞系RAW264.7抗绵羊肺炎支原体(Mycoplasma ovipneumoniae,MO)感染中的作用,实验利用CCK-8细胞活性检测找到最佳甘草酸处理浓度;检测甘草酸对受MO感染的巨噬细胞活性的影响。流式细胞仪检测甘草酸对RAW264.7巨噬细胞生长周期的影响;ELISA检测甘草酸对受MO感染的巨噬细胞分泌TNF-α的影响;Western blot检测细胞凋亡因子Bax、Bad的表达情况。RT-PCR检测凋亡和自噬相关基因的表达情况。结果显示,浓度为12μmol/L的甘草酸显著升高RAW264.7的活性(P=0.012 9),且处于G1期的细胞数减少,G2期的细胞数增加。甘草酸(12μmol/L)可提高受MO感染的RAW264.7的增殖率(P=0.034 0),培养上清中TNF-α含量升高(P=0.015 2),巨噬细胞中促凋亡蛋白Bax表达量增加,但基因caspase 3和caspase 9的表达量显著下调(P<0.000 1),自噬相关基因Atg 7和Beclin 1表达量显著升高(P<0.000 1)。结果提示在MO感染巨噬细胞引起免疫抑制的情况下,甘草酸可通过促增殖、抑凋亡、促进TNF-α的表达、增加自噬来起到免疫调控作用。     

流感病毒感染巨噬细胞对缺氧诱导因子-1α诱导炎症反应通路的机制研究

为探索缺氧诱导因子(hypoxia inducible factor,HIF)-1α诱导甲型流感病毒毒株感染小鼠巨噬细胞引起炎症反应的具体机制,本研究以甲型H1N1流感病毒(简称H1N1)株A/PR/8感染小鼠巨噬细胞RAW264.7后,在显微镜下观察其在感染后的表型变化,分别在不同时间段收集样本,通过聚合酶链反应(polymerase chain reaction,PCR)检测HIF-1α、干扰素(interferon,IFN)-γ、白细胞介素(interleukin,IL)-6、肿瘤坏死因子(tumor necrosis factor,TNF)-α和M蛋白(M protein,MBP)mRNA的变化,通过蛋白质印迹法(Western blot, WB)检测HIF-1α、核转录因子-κB(nuclear factor-κB,NF-κB)、促分裂原活化的蛋白激酶(mitogenactivated protein kinase,MAPK)、蛋白激酶B(protein kinase B,Akt)以及M蛋白的变化。随后,加入抑制剂2-MeOE-2(10 nmol/L)进行抑制试验,采用PCR和WB检测HIF-1α表达被抑制后上述炎症因子mRNA表达水平及炎症蛋白通路的变化。结果显示,H1N1PR8感染小鼠巨噬细胞RAW264.7后,H1N1PR8复制率在24 h达到峰值,HIF-1α mRNA在感染6 h后开始升高,12 h迅速上升,24 h达到峰值。IFN-γ、IL-6、TNF-α mRNA变化趋势基本与HIF-1α一致,但在感染12 h并未进入快速上升期。HIF-1α蛋白在感染后6 h表达明显增多,24 h达到峰值,与mRNA变化水平基本一致。NF-κB通路蛋白在感染12 h后明显增多,48 h开始减少。加入抑制剂2-MeOE-2后,培养感染细胞24 h,抑制剂组IL-6、TNF-α mRNA水平较对照组显著下降(P<0.05),抑制剂组NF-κB通路蛋白较对照组表达下降。本研究结果表明,小鼠巨噬细胞被H1N1感染后,HIF-1α可能通过激活NF-κB通路促进IL-6、TNF-α等炎症因子的分泌参与炎症反应。     

艾拉莫德(T-614)对巨噬细胞M1型极化的影响

[目的]研究艾拉莫德(T-614)对小鼠巨噬细胞(RAW264.7)M1型极化的影响。[方法]细胞毒性实验观察3个浓度(400 g/L,800 g/L,1 200 g/L)的T-614对RAW264.7的影响,使用LPS/IFN-γ诱导RAW264.7发生M1型分化,同时进行T-614干预。流式细胞术检测RAW264.7表面F4/80+CD86+与MHCⅡ+的比例,ELISA检测细胞中IL-1β、IL-6、TNF-α的含量,RT-PCR检测细胞中IL-1β、IL-6、TNF-α、MCP-1、CD86和iNOS基因的表达,Western Blot检测细胞中MCP-1、CD86和iNOS蛋白表达水平。[结果]3个浓度T-614对未分化的巨噬细胞没有毒性;高浓度T-614降低M1巨噬细胞表面的F4/80+CD86+与MHCⅡ+比例(P<0.05),降低MCP-1、CD86和iNOS的基因表达水平与蛋白表达水平(P<0.05),降低IL-1β、IL-6、TNF-α基因表达与减少IL-1β、IL-6、TNF-α的含量(P<0.05)。[结论]T-614能抑制RAW264.7进行M1型极化,抑制MCP-1、CD86和iNOS的表达,减少IL-1β、IL-6、TNF-α的形成与分泌。     

结核分枝杆菌Rv2626c诱导的细胞免疫应答特性

全球有近1/4的人感染结核分枝杆菌(Mycobacterium tuberculosis,M.tb)并长期处于潜伏感染状态。Rv2626c是结核分枝杆菌受DosR调控的重要潜伏感染相关蛋白。本研究对Rv2626c蛋白进行了原核表达和纯化,并以RAW264.7细胞和小鼠为感染模型,对其免疫生物学特性进行了分析。SDS-PAGE及Western blotting鉴定结果表明,Rv2626c-His融合蛋白主要以可溶形式表达,能与兔抗H37Rv多抗血清发生特异性免疫反应。此外,本研究发现Rv2626c蛋白能结合到巨噬细胞RAW 264.7表面并上调细胞NO的生成;显著诱导促炎细胞因子IFN-γ、TNF-α、IL-6和MCP-1的产生;并能诱导小鼠产生更强的Th1免疫应答。上述研究有利于揭示结核分枝杆菌的致病机制,为新型结核病疫苗的研制奠定了理论基础。     

趋化因子MCP-1、MIP-1α的表达与TAM计数在胆囊癌中的临床病理意义

目的：检测胆囊腺癌组织中趋化因子MCP-1和MIP-1α的表达、TAM计数并探讨其临床病理意义。方法：收集中南大学湘雅二医院及湖南省人民医院近五年胆囊腺癌手术切除标本36例及慢性胆囊炎手术切除标本10例,采用原位分子杂交方法检测MCP-1和MIP-1α的表达,免疫组化法进行TAM计数。结果：胆囊腺癌组织中MCP-1、MIP-1αmRNA表达阳性率及评分均明显高于慢性胆囊炎（P〈0．01）;高分化胆囊腺癌中二者的阳性率及评分均低于低分化胆囊腺癌,其中MCP-1mRNA比较有显著性差异（P〈0．05）;MCP-1、MIP-1α mRNA的表达呈显著正相关。胆囊腺癌组织MCP-1mRNA表达阳性率及其评分与侵犯胆总管及发生淋巴结转移显著相关;MIP-1α mRNA表达阳性率及其评分与侵犯肝脏显著相关。胆囊腺癌组织中,TAM计数（24．89±0．84）明显高于慢性胆囊炎组织（16．19±0．66）,有显著性差异（P〈0．01）。TAM与MCP-1、MIP-1α mRNA表达评分值均呈显著正相关（r分别为0．580,0．567）。MCP-1 mRNA与MIP-1α mRNA评分值之间呈显著正相关（r=0．638）。结论：MCP-1、MIP-1α的表达增加及TAM计数升高可能调控和影响胆囊癌的发生和发展,MCP-1、MIP-1α可能促进TAM向胆囊癌组织迁移浸润。     

趋化因子MIP-2和MCP-1在结直肠癌中表达及临床病理意义

目的:观察结直肠癌组织中趋化因子巨噬细胞炎性蛋白-2(MIP-2)和单核细胞趋化蛋白-1(MCP-1)蛋白的表达,分析其表达的临床病理意义.方法:收集结直肠癌病例的病理资料和临床资料,应用免疫组织化学SP法检测待测蛋白的表达.结果:MIP-2在癌旁肠组织组和腺癌组的阳性表达率分别为76%,68%,差异无统计学意义.MCP-1在癌旁肠组织组和腺癌组的阳性表达率分别为56%,72%,差异无统计学意义.MIP-2表达与淋巴结转移有关,而与肿块大小、部位、分化程度、浸润肠壁深度无关.MCP-1表达与肿块直径、浸润肠壁深度、淋巴结转移有关,而与部位、分化程度无关.结论:MIP-2和MCP-1在结直肠癌中的表达主要与侵袭和转移相关.     

趋化因子MCP-1、MIP-1alpha的表达与TAM 计数在胆囊癌中的 临床病理意义

目的：检测胆囊腺癌组织中趋化因子MCP-1 和MIP-1-alpha的表达、TAM 计数并探讨其临床病理意义。方法：收集中南大学湘 雅二医院及湖南省人民医院近五年胆囊腺癌手术切除标本36 例及慢性胆囊炎手术切除标本10 例,采用原位分子杂交方法检测 MCP-1 和MIP-1alpha的表达,免疫组化法进行TAM计数。结果：胆囊腺癌组织中MCP-1、MIP-1alpha mRNA表达阳性率及评分均明显 高于慢性胆囊炎(P<0.01);高分化胆囊腺癌中二者的阳性率及评分均低于低分化胆囊腺癌,其中MCP-1 mRNA 比较有显著性差异 (P＜0.05);MCP-1、MIP-1-alpha-mRNA的表达呈显著正相关。胆囊腺癌组织MCP-1 mRNA表达阳性率及其评分与侵犯胆总管及发生 淋巴结转移显著相关;MIP-1alpha-mRNA表达阳性率及其评分与侵犯肝脏显著相关。胆囊腺癌组织中,TAM 计数(24.89± 0.84)明显 高于慢性胆囊炎组织(16.19± 0.66),有显著性差异(P<0.01)。TAM与MCP-1、MIP-1alpha mRNA 表达评分值均呈显著正相关(r 分别为 0.580,0.567)。MCP-1mRNA 与MIP-1alpha-mRNA评分值之间呈显著正相关(r＝0.638)。结论：MCP-1、MIP-1alpha的表达增加及TAM 计 数升高可能调控和影响胆囊癌的发生和发展,MCP-1、MIP-1琢可能促进TAM向胆囊癌组织迁移浸润。     

Increased expression of B-lymphocyte chemoattractant,but not pro-inflammatory cytokines,in muscle tissue in rhesus chronic Lyme borreliosis

Inflammation in skeletal muscle is a consistent feature of Lyme borreliosis, both in the human disease and experimental models. This study had two goals: to evaluate the expression of selected pro-inflammatory and chemokine genes in skeletal muscle in the Rhesus model of Lyme disease, and to identify unexpected cytokine genes involved in Lyme myositis. Two different techniques for measuring cytokine messenger RNA (mRNA) levels were used to achieve these goals: gene expression microarrays and. real-time RT-PCR (Taqman). Muscle from necropsies and biopsies were used, and were obtained from both infected and uninfected non-human primates (NHPs). Although many cytokines were found expressed in muscle tissue, pro-inflammatory cytokines commonly associated with inflammation were not consistently upregulated in infected muscles relative to uninfected muscles. However, B-lymphocyte chemoattractant (BLC), a chemokine implicated in the trafficking of B-cells into tissue, was increased in expression. This study is the first to extensively characterize cytokine gene expression in chronically inflamed tissue in Lyme borreliosis.     

Changes in gene expression induced by H(2)O(2) in cardiac myocytes

Oxidative stress induces cardiac myocyte apoptosis. At least some effects are probably mediated through changes in gene expression. Using Affymetrix arrays, we examined the changes in gene expression induced by H(2)O(2) (0.04, 0.1, and 0.2mM; 2 and 4h) in rat neonatal ventricular myocytes. Changes in selected upregulated genes were confirmed by ratiometric RT-PCR. p21(Cip1/Waf1) was one of the only two genes upregulated in all conditions studied. Of the heat shock proteins, only Hsp70/70.1 was induced by H(2)O(2) with no change in the expression of Hsp25, Hsp60 or Hsp90. Heme oxygenase 1 was also potently upregulated, but not heme oxygenases 2 or 3. Of the intercellular adhesion proteins, syndecan-1 was significantly upregulated in response to H(2)O(2), with little change in the expression of other syndecans and no change in expression of any of the integrins studied. Thus, oxidative stress, exemplified by H(2)O(2), selectively promotes the expression of specific gene family members.     

Acinetobacter baumannii Outer Membrane Vesicles Elicit a Potent Innate Immune Response via Membrane Proteins

Acinetobacter baumannii is increasingly becoming a major nosocomial pathogen. This opportunistic pathogen secretes outer membrane vesicles (OMVs) that interact with host cells. The aim of this study was to investigate the ability of A. baumannii OMVs to elicit a pro-inflammatory response in vitro and the immunopathology in response to A. baumannii OMVs in vivo. OMVs derived from A. baumannii ATCC 19606^T induced expression of pro-inflammatory cytokine genes, interleukin (IL)-1β and IL-6, and chemokine genes, IL-8, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1, in epithelial cells in a dose-dependent manner. Disintegration of OMV membrane with ethylenediaminetetraacetic acid resulted in low expression of pro-inflammatory cytokine genes, as compared with the response to intact OMVs. In addition, proteinase K-treated A. baumannii OMVs did not induce significant increase in expression of pro-inflammatory cytokine genes above the basal level, suggesting that the surface-exposed membrane proteins in intact OMVs are responsible for pro-inflammatory response. Early inflammatory processes, such as vacuolization and detachment of epithelial cells and neutrophilic infiltration, were clearly observed in lungs of mice injected with A. baumannii OMVs. Our data demonstrate that OMVs produced by A. baumannii elicit a potent innate immune response, which may contribute to immunopathology of the infected host.     

Proinflammatory Cytokine Response and Viral Replication in Mouse Bone Marrow Derived Macrophages Infected with Influenza H1N1 and H5N1 Viruses

The pathogenesis of human influenza H5N1 virus infection remains poorly understood and controversial. Cytokine dysregulation in human infection has been hypothesized to contribute to disease severity. We developed in vitro cultures of mouse bone marrow derived macrophages (BMDMΦ) from C57BL/6N mouse to compare influenza A (H5N1 and H1N1) virus replication and pro-inflammatory cytokine and chemokine responses. While both H1N1 and H5N1 viruses infected the mouse bone marrow derived macrophages, only the H1N1 virus had showed evidence of productive viral replication from the infected cells. In comparison with human seasonal influenza H1N1 (A/HK/54/98) and mouse adapted influenza H1N1 (A/WSN/33) viruses, the highly pathogenic influenza H5N1 virus (A/HK/483/97) was a more potent inducer of the chemokine, CXCL 10 (IP-10), while there was not a clear differential TNF-α protein expression pattern. Although human influenza viruses rarely cause infection in mice without prior adaption, the use of in vitro cell cultures of primary mouse cells is of interest, especially given the availability of gene-defective (knock-out) mice for specific genes.