内吗啡肽对小鼠树突状细胞免疫功能的影响

目的研究内吗啡肽-1和内吗啡肽-2对小鼠骨髓来源树突状细胞表型和免疫功能的影响。方法从7～8周龄的C57BL/6J小鼠提取骨髓前体细胞培养,经CD11c免疫磁珠分选,获得纯化的树突状细胞(dendritic cells,DC)。未成熟DC用脂多糖或脂多糖+不同浓度内吗啡肽共培养,流式细胞术检测DC的表型;检测内吗啡肽对DC趋化性的影响。在DC和T淋巴细胞共培养的条件下,用氚标记胸腺嘧啶核苷(3H-TdR)参入测定的方法检测T淋巴细胞的增殖能力。结果内吗啡肽抑制DC的趋化性;与T淋巴细胞体外共培养时,经内吗啡肽-1或内吗啡肽-2处理的DC能抑制T淋巴细胞的增殖反应。上述内吗啡肽的作用都能被Mu阿片受体特异性拮抗剂CTOP翻转或部分翻转,提示内吗啡肽的作用是由Mu受体介导的。结论内吗啡肽可通过作用于Mu阿片受体,改变树突状细胞的部分免疫功能,调节免疫反应。     

内吗啡肽-1对人树突状细胞TLR2和TLR4表达的影响

目的观察内吗啡肽-1(EM-1)对人外周血来源树突状细胞TLR2和TLR4表达的影响。方法从人外周血中提取和分离单核细胞,在含重组人粒-巨噬细胞集落刺激因子和重组人白细胞介素-4的完全培养基中培养,6 d后未成熟树突状细胞(im DC)分4组,空白对照组(BLA组)、EM-1组、LPS组、LPS+EM-1组。继续培养2 d后,流式细胞术检测各组DC表面TLR2、TLR4蛋白的表达;RT-PCR法检测各组DC中TLR2 mRNA、TLR4 mRNA的表达。结果流式结果显示,TLR2、TLR 4在im DC表达较高,随着DC成熟表达下降。与BLA组相比,EM-1组DC表面TLR2、TLR4的表达下调(P<0.05);与LPS组相比,LPS+EM-1组DC表面TLR2、TLR4受体均明显下调(P<0.01)。RT-PCR的结果显示,与BLA组相比,EM-1干预后引起DC表面TLR2 mRNA表达明显降低(P<0.01),TLR4 mRNA的变化无差异(P>0.05);与LPS组相比,LPS+EM-1组DC表面TLR2 mRNA、TLR4mRNA均有所下调(P<0.05)。结论 EM-1使DC表面TLR2、TLR4的表达下调,EM-1对DC免疫功能的影响可能与DC表面TLR2、TLR4表达有关。     

内源性阿片样肽——内吗啡肽的研究进展

阿片肽的研究已有30年的历史。1997年，又发现了μ阿片受体的高选择性、高亲和性的内源性配体——内吗啡肽(EM)，它通过与G蛋白偶联的μ阿片受体结合从而发挥许多生物学效应。本文就近年来EM的研究进展，特别是关于它在中枢神经系统内分布、受体结合特性、镇痛作用的特点及其机制，以及对心血管、呼吸、消化功能的调节等方面作一综述。     

内吗啡肽-1对麻醉大鼠左心室功能的影响

目的：观察内吗啡肽-1（endomorphin-1,EM-1）静脉注射和侧脑室注射对麻醉大鼠左心室功能的影响,并初步探讨其可能的作用机理。方法：Wistar大鼠麻醉后,静脉注射或侧脑室注射EM-1,经右颈总动脉左心室插管测定左心室功能,并测定血清和心肌组织NO和T-NoS含量。结果：静脉注射、侧脑室注射EM-1均剂量依赖性地降低麻醉大鼠左心室功能,预先给予阿片受体阻断剂纳洛酮或特异性肚受体阻断剂cyprodime可阻断EM-1引起的心功能降低反应;预先给予胆碱能M受体阻断剂阿托品可减弱EM-1引起的心功能降低反应。静脉注射EM-1血清中NO和T-NOS比对照组有所增加,而心肌组织的NO和T-NOS无明显变化;侧脑室注射EM-1的血清和心肌组织的NO和T-NOS均无明显变化。结论：静脉注射、侧脑室注射EM-1可引起麻醉大鼠在体左心室功能下降,此效应由阿片受体介导,可能有胆碱能M受体参加。     

内吗啡肽研究进展

1997年发现的内吗啡肽 (Endomorphins、EMs) ,结构上属于四肽 ,被认为是 μ阿片受体 (MOR)的内源性配基 ,它通过与G蛋白偶联的MOR受体结合介导许多生理活动 ,本文结合本实验室对内吗啡肽的研究 ,对其神经系统分布、受体结合特性、生理作用、构象及构效关系等方面进行介绍     

侧脑室注射内吗啡肽-1对麻醉大鼠血压的影响

目的　观察侧脑室注射内吗啡肽-1(EM-1)对麻醉大鼠血压的影响,并初步探讨其作用机理。方法　侧脑室埋植导管给药,颈动脉插管测血压。结果　icv EM-1剂量依赖、纳洛酮敏感地降低麻醉大鼠的血压。icv或iv酚妥拉明、普萘洛尔和iv L-NNA对EM-1引起的血压降低反应无影响;给予阿托品(icv 25 μg·kg^-1 ;或iv 50 μg·kg^-1)和切断双侧迷走神经减弱EM-1引起的血压降低反应。结论　icv EM-1可引起麻醉大鼠血压降低;此效应由阿片受体介导,有中枢M受体的参与,通过兴奋迷走神经所致     

内吗啡肽—1的镇痛作用

目的：研究内吗啡肽－1（EM－1）的镇痛作用。方法：采用电刺激鼠尾－嘶叫法、扭体法、佐剂性关节炎以及神经源性疼痛等多种疼痛模型,观察腹腔注射EM－1的镇痛作用,并和脊髓蛛网膜下腔注射和侧脑室注射EM－1的镇痛作用进行比较。结果：1）EM－1能剂量依赖地提高大鼠电刺激鼠尾－嘶叫法的痛阈;能抑制醋酸引起的小鼠扭体反应;在佐剂性关节炎所致的炎症性痛觉过敏及坐骨神经部分结扎所引起的神经源性痛觉过敏中,EM－1与有镇痛作用。2）中枢给EM－1的镇痛作用比外击给药出现得较快,而且较强。3）阿片受体拮抗选择性拮抗剂cyprodime也能翻转EM－1的镇痛作用;反复给予EM－1具有确切的镇冯作用,其镇痛作用由中枢μ阿片受体介导。     

吗啡对体外培养的人乳腺细胞雌激素受体mRNA表达的影响

目的：研究吗啡对雌激素诱导的体外培养人的正常乳腺细胞的雌激素受体（ER）mRNA表达的影响。方法：购买人正常乳腺纤维细胞株进行培养，给予17-β雌二醇（E2），吗啡（Mor）及纳洛酮干预。通过RT—PCR的方法半定量分析吗啡对正常乳腺细胞ER mRNA表达的影响。结果：ER与内参照B—actin分别在249 bp和270 bp处出现特征性条带。雌激素组、吗啡组与对照组比较，差异有显著性（P〈0．05），E2＋Mor组与对照组比较，差异无显著性（P〉0．05）。吗啡作用24h呈剂量依赖性抑制乳腺细胞ERmRNA的表达，3组吗啡的终浓度分别为1×10^-6mol／L、1×10^-5mol／L、1×10^-4 mol／L，差异有显著性（P〈0，05）。结论：从mRNA水平证明了外源性阿片类药物吗啡可直接影响雌激素对乳腺的促增殖作用，同时还可剂量依赖性下调雌激素受体mRNA的表达。     

绝经后妇女长期低剂量激素替代疗法中外周血内吗啡肽1和2的分析研究

目的在长期低剂量激素替代疗法(HRT)下,观察绝经后妇女外周血中内吗啡肽1(EM-1)和内吗啡肽2(EM-2)的变化,并对内吗啡呔与雌激素进行相关性分析。方法研究对象系北京协和医院已绝经女职工共169人,全员根据年龄共分为4组,包括50~59岁,60~64岁,65~69岁,70岁以上,每年龄段内均包括HRT组和对照组。用酶联免疫反应测试仪测定受试者血浆性激素水平,用放射免疫法检测血浆内EM-1和EM-2的含量。结果各年龄段HRT治疗组E2的水平均显著高于对照组(P<0.05);各年龄段HRT治疗组EM-1和EM-2的水平均低于对照组,且全体受试者血浆雌激素E2的水平与EM-1具有Pearson’s相关性。结论首次观察了在长期低剂量HRT疗法下绝经后妇女外周血中内吗啡肽1和2的变化,该结果对如何保护绝经后妇女的脑认知具有一定的临床意义。     

混合酸酐法合成内吗啡肽-1

目的:采用液相方法合成内吗啡肽-1.方法:以混合酸酐法先分别合成Ⅳ端二肽片段和C端二肽片段,再缩合得到四肽,以50%TFA/DCM(三氟乙酸/二氯甲烷)脱除Ⅳ端Boc(叔丁氧羰基)保护基.结果:合成的内吗啡肽-1(EM-1),收率较文献高,结构经质谱和1H NMR确证.结论:混合酸酐法操作简便,反应时间缩短,副产物少,易分离纯化,适用于大量制备.     

Analysis of poly(D,L-lactic-co-glycolic acid) nanosphere uptake by human dendritic cells and macrophages in vitro

Purpose. The purpose of this study was to demonstrate and characterize phagocytosis of poly(D,L-lactic-co-glycolic acid) (PLGA) nanospheres by human dendritic cells (DCs). Methods. Parallel cultures of DCs and macrophages (M) were established from peripheral blood leukocytes using media supplemented with granulocyte-macrophage colony stimulator factor and interleukin-4 (for DC) or granulocyte-macrophage colony stimulator factor alone (for M). PLGA nanospheres containing tetramethylrhodamine-labeled dextran with or without an adjuvant, monophosphoryl lipid A, were prepared using a water/oil/water solvent evaporation technique. Cells were incubated with the nanospheres for 24 h. Confocal laser scanning microscopy was used to determine the intracellular location of the nanospheres and flow cytometry to measure the fraction of phagocytic cells in the culture and the amount of uptake per cell. After phagocytosis, cells were stained for MHC class II molecules, CD14, CD80, and CD86 to identify the phagocytic population. Results. DCs phagocytosed PLGA nanospheres as efficiently as M. Cell-surface marker expression conclusively established that the phagocytic cells were DC. Conclusions. DCs can take up PLGA nanospheres. Because DCs are the key professional antigen-presenting cells capable of stimulating naive T cells, our data suggest that PLGA nanospheres can be used as an efficient delivery system for vaccines designed to activate T cell-mediated immune responses.     

Developing dendritic cell-based therapies to condition immune responses to novel oncogenic proteins and stem cells

Cancer vaccines have been disappointing when utilized as stand-alone therapy, especially in late disease settings. However, recent clinical studies in prostate cancer have suggested that dendritic cellular (DC) vaccines may impact patient survival, reviving the notion that cancer vaccines can impact established cancer. In this review we will highlight the advances that have been made in the development of DC-based therapies activated by Toll-like receptor agonists with the capacity to condition toward strong Th1 cellular responses, through the production of cytokines and chemokines, and a capacity to induce apoptosis of tumor cells. Used in early cancer settings, these DCs induce clinically effective immune responses, thus shifting the emphasis toward using these cells earlier in the disease process. We will also discuss targeting novel molecules and cancer stem cells that can eliminate cells with high metastatic potential, moving DC-based therapies into mainstream cancer therapy.     

1,25-二羟维生素D3对其诱导的树突状细胞上Toll样受体7表达的抑制作用

目的探讨Toll样受体7（TLR7）在1,25-二羟维生素D3（1,25-（OH）2D3）抑制树突状细胞（DC）成熟中可能的机制。方法体外扩增小鼠骨髓来源的DC,采用流式细胞术和混合淋巴细胞反应检测1,25-（OH）₂D₃对DC表型和功能的影响;利用RT-PCR半定量方法测定1,25-（OH）₂D₃对DC的TLR7表达的影响。结果 1,25-（OH）₂D₃处理后,DC表达CD86和MHCⅡ的荧光强度均明显降低;对T细胞的刺激能力较对照组明显减弱,差异有统计学意义（P<0.05）;与对照组比较,1,25-（OH）₂D₃可以明显下调TLR7的表达（P<0.05）。结论 1,25-（OH）₂D₃可能通过影响TLR7的表达来抑制DC细胞的成熟,从而使DC具有耐受性特征。     

Cigarette smoke suppresses TLR-7 stimulation in response to virus infection in plasmacytoid dendritic cells

Exposure to environmental tobacco smoke (ETS) is associated with an increase in the frequency and severity of respiratory infections, including bronchiolitis, a clinical syndrome of infancy caused by viruses such as respiratory syncytial virus (RSV). The mechanisms by which ETS increases the risk of viral respiratory infections are largely unknown. A major effector integrating early antiviral and immunostimulatory activities is interferon-α (IFN-α), which is highly produced by plasmacytoid dendritic cells (pDC). In this work, we determined the effect of cigarette smoke extract (CSE) on human pDC immunity in response to a respiratory viral infection. We found that CSE inhibited RSV-induced IFN-α in pDC as well as the release of IL-1β, IL-10 and CXCL10. However, the production of additional cytokines and chemokines such as IL-6, TNF-α, CCL2, CCL3, CCL5 and CXCL8 was not altered. Quantitative RT-PCR analysis indicated that CSE decreased the expression of toll-like receptor (TLR)-7 and interferon regulatory factor (IRF)-7 in RSV-infected pDC. Furthermore, determination of IRF-7 phosphorylation by flow cytometry showed that CSE prevented IRF-7 activation. These data provide evidence that cigarette smoke suppresses key pDC functions upon viral infection by a mechanism that involves downregulation of TLR7 expression and decreased activation of IRF-7.     

Loxoribine,a selective Toll-like receptor 7 agonist,induces maturation of human monocyte-derived dendritic cells and stimulates their Th-1- and Th-17-polarizing capability

Recently, a guanosine analog, 7-allyl-7,8-dihydro-8-oxo-guanosine (loxoribine), has been identified as a selective Toll-like receptor (TLR)7 agonist. Bearing in mind the controversy regarding the expression of TLR7 by human myeloid dendritic cells (DCs) and its significance for functions of these cells, the goal of this study was to investigate the effect of loxoribine on differentiation, maturation and functions of human monocyte-derived (Mo)DCs. Immature MoDCs were obtained by cultivation of monocytes for 6 days with recombinant granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4. These cells were stimulated with loxoribine (250 μM) for an additional 48 h. Phenotypic properties of MoDCs were determined by flow cytometry, cytokine production was assayed by ELISA, whereas their allostimulatory capability was tested using a mixed leukocyte reaction. We showed that loxoribine up-regulated the expression of TLR7, CD40, CD54, CD80, CD83 and CCR7 and stimulated the production of IL-12, IL-23, IL-27 and IL-10 by MoDCs, whereas the level of interferon (IFN)-β was not modulated. Allogeneic CD4⁺T cells in co-culture with loxoribine-treated MoDCs proliferated more strongly, at lower DC/CD4⁺T-cell ratio (1:80), and secreted significantly higher levels of IL-17 and IFN-γ compared to the cultures with control MoDCs. The stimulatory effect of loxoribine on T helper (Th)1 polarization capability of MoDCs was further potentiated by ligation of CD40. In conclusion, our results show that loxoribine stimulated differentiation, maturation, allostimulatory as well as Th1 and Th17 polarization capability of human MoDCs and suggests that these effects might be associated with up-regulation of TLR7 expression, but not increased IFN-β production.     

Inducible Expression of Functional Mu Opioid Receptors in Murine Dendritic Cells

Opioids are known to exert direct effects on the immune system, and the expression of functional opioid receptors has been reported on several immune cell types. Dendritic cells (DCs) are important inducers and regulators of immune responses. In this study, we investigated whether murine dendritic cells express functional mu opioid receptors (MOR). RT-PCR analysis and double immunofluorescence staining revealed the expression of MOR in activated murine dendritic cells. We also studied the dynamic expression of MOR messenger RNA in murine dendritic cells in response to different Toll-like receptor ligands. Functionally, treatment of DCs with endomorphin 1 (EM1), a specific agonist of MOR, can inhibit the forskolin-induced formation of cyclic adenosine monophosphate level in activated DCs. Moreover, EM1 treatment resulted in less activation of p38 MAPK and more activation of ERK signaling in lipopolysaccharide-stimulated DCs. Consistently, treatment of DCs with EM1 altered cytokine production by increasing IL-10 and decreasing IL-12 and IL-23. Our results suggest that MOR is inducibly expressed on activated DCs and functionally mediates EM1-induced effects on DCs. Thus, dendritic cells might be involved in crosstalk between the neuroendocrine and the immune system.     

Activation of the inflammasome by amorphous silica and TiO2 nanoparticles in murine dendritic cells

Abstract

Nanomaterials are increasingly used in various food applications. In particular, nanoparticulate amorphous SiO₂ is already contained, e.g., in spices. Since intestinal dendritic cells (DC) could be critical targets for ingested particles, we compared the in vitro effects of amorphous silica nanoparticles with fine crystalline silica, and micron-sized with nano-sized TiO₂ particles on DC. TiO₂- and SiO₂-nanoparticles, as well as crystalline silica led to an upregulation of MHC-II, CD80, and CD86 on DC. Furthermore, these particles activated the inflammasome, leading to significant IL-1β-secretion in wild-type (WT) but not Caspase-1- or NLRP3-deficient mice. Silica nanoparticles and crystalline silica induced apoptosis, while TiO₂ nanoparticles led to enhanced production of reactive oxygen species (ROS). Since amorphous silica and TiO₂ nanoparticles had strong effects on the activation-status of DC, we suggest that nanoparticles, used as food additives, should be intensively studied in vitro and in vivo, to ensure their safety for the consumer.     

布鲁氏菌病患者外周血树突状细胞、Th1／Th2细胞因子含量的检测及意义

目的：观察布鲁氏菌病患者外周血树突状细胞表型、Th1／Th2细胞含量的检测及意义。方法选取诊治的布鲁氏菌病患者50例为病例组,另选取同期进行健康体检的正常人50例作为正常组。采用Real time-PCR测定2组Th1相关转录因子T细胞表达的T盒（T-bet）、GATA连接蛋白3（GATA-3）、维A酸相关核孤儿受体γt（RORγt）、叉头蛋白3（Foxp3）及Th1／Th2细胞因子肿瘤坏死因子α（TNF-α）、干扰素γ（IFN-γ）、白介素6（IL-6）、白介素10（IL-10）mRNA含量。酶联免疫吸附实验（ELISA）测定TNF-α、IFN-γ、IL-6、IL-10蛋白表达及补体C3、C4含量。流式细胞术测定树突状细胞表型、Th1、Th2及T淋巴细胞亚群（ CD4＋T细胞、CD8＋T细胞、NK细胞）含量。结果病例组外周血Th1细胞相关转录因子T-bet、RORγt、Foxp3及Th1细胞、Th1／Th2、TNF-α、IFN-γ含量较对照组显著降低,Th2细胞及IL-6、IL-10含量较对照组显著升高,差异有统计学意义（ P ＜0．05）;病例组CD8＋3、CD8＋0、CD8＋6阳性的树突状细胞比例、CD4＋T细胞、NK细胞及补体C3、C4含量较对照组显著降低,CD8＋T细胞含量较对照组显著升高,差异有统计学意义（ P ＜0．05）;TNF-α、IFN-γ含量与CD4＋T细胞、NK细胞、C3、C4含量呈正相关性（ r值分别为2．879、3．214、3．255和2．978, P ＜0．05）,与CD8＋T细胞含量呈负相关性（ r值分别为－3．146和－3．011, P ＜0．05）。 IL-6、IL-10含量与CD4＋T细胞、NK细胞、C3、C4含量呈负相关性（ r值分别为－2．124、－2．343、－3．423、－2．789、－2．993、－2．566、－3．758, P ＜0．05）,与CD8＋T细胞含量呈正相关性（ r值分别为3．465、3．129, P ＜0．05）。结论布鲁氏菌病患者外周血成熟树突状细胞数目减少,同时Th1／Th2细胞及相关细胞因子失衡,且与机体天然免疫和细胞免疫功能降低有关,这可能在布鲁氏菌病发生发展过程中发挥重要作用。