MyD88 RNA干扰抑制小鼠骨髓树突状细胞成熟的实验研究

目的：针对小鼠骨髓树突状细胞（DC）髓性分化因子88（MyD88）,采用RNA干扰技术抑制其合成,观察脂多糖（LPS）刺激后DC生物学活性的变化,探讨获得耐受性DC的方法,为DC的临床应用提供新思路和理论依据。方法：培养小鼠骨髓源性DC,分为对照组、LPS组及RNA干扰组,对照组不予任何处理,LPS组加入终浓度为1μg/ml的LPS,RNA干扰组于加入MyD88 siRNA12小时后给予1μg/mlLPS刺激,继续培养3天。免疫细胞化学检测DCMyD88、核因子-κB（NF-κB）的表达,Western blot检测DCMyD88的表达;流式细胞术检测DC细胞表面CD80、CD86及MHC-Ⅱ分子的变化,ELISA法检测各组DC分泌TNF-α。IFN-γ和IL-12的浓度,混合淋巴细胞培养检测T细胞增殖能力。结果：LPS促进DC高表达CD80、CD86及MHC-Ⅱ分子,促进Th1型细胞因子释放、MyD88高表达及NF-κB核移位,并诱导T细胞增殖,MyD88 siRNA可阻断LPS的这些效应。结论：MyD88 siRNA可抑制DC成熟,产生耐受性DC,增强同种未成熟DC的免疫耐受诱导作用,为进一步研究DC的临床应用奠定了基础。     

林可霉素对树突状细胞系DC2.4免疫功能影响的初步研究

目的:探讨林可霉素(lin)对树突状细胞(DCs)系DC2.4免疫功能的影响。方法:设立3个组:DC2.4细胞组,DC2.4细胞+LPS组及DC2.4细胞+LPS+lin组(LPS及lin均为500 ng/mL)。在倒置显微镜下观察各实验组中DC2.4细胞的形态学变化。同时采用流式细胞仪分析DC2.4细胞表面标志MHC-Ⅱ类分子、CD86和CD80的表达。采用同种异体混合淋巴细胞反应(MLR)检测各实验组中DC2.4细胞刺激同种异体T淋巴细胞增殖的能力。用ELISA试剂盒检测干扰素-γ(IFN-γ)水平的变化。结果:倒置显微镜下显示,DC2.4细胞组为未成熟DCs形态,DC2.4细胞+LPS组及DC2.4细胞+LPS+lin两组均为成熟DCs的形态。流式细胞术分析证实,500 ng/mL LPS可以促进DC2.4细胞表面MHC-Ⅱ类分子、CD86和CD80的表达,并增强其刺激T细胞增殖及IFN-γ的分泌。但500 ng/mL lin联合500 ng/mL LPS能够部分抑制DC2.4细胞免疫调节作用。结论:Lin可以部分抑制成熟DC2.4细胞的免疫调节功能。     

HBeAg对小鼠骨髓源性树突状细胞成熟的影响

目的 探讨HBe Ag对LPS诱导小鼠骨髓源性树突状细胞(DC)成熟的影响。方法 体外诱导C57BL/6小鼠骨髓细胞分化成未成熟树突状细胞,经CD11c磁珠分选纯化后将DCs随机分为空白对照组、LPS刺激组、HBe Ag+LPS刺激组。流式检测DC表型变化,混合淋巴反应(MLR)检测DC促T淋巴细胞增殖能力,酶联免疫法(ELISA)检测细胞上清液中IL-12的分泌水平,Western blot检测p38磷酸化水平,并设置SB203580组为阳性对照探讨细胞IL-12分泌的可能调节机制。结果 LPS刺激未成熟DC引起细胞表面MHC-Ⅱ、CD86表达升高,刺激同种异体淋巴细胞增殖能力增强,IL-12分泌量增高。HBe Ag可抑制LPS促进DC表面MHC-Ⅱ、CD86表达升高和促淋巴细胞增殖能力增强的作用。LPS刺激DC可引起p38磷酸化水平升高,并呈时间依赖性;HBe Ag或SB203580预处理细胞再予LPS刺激,磷酸化p38表达和IL-12分泌较单纯LPS刺激组明显下降。结论 HBe Ag对LPS引起的树突状细胞的成熟有一定的抑制作用,且HBe Ag可能通过抑制p38MAPK信号通路下调LPS诱导的树突状细胞IL-12的产生。     

补体调节蛋白Crry对树突状细胞的调控及诱导同种移植免疫低反应

目的:探讨补体调节蛋白Crry对树突状细胞(DC)的调节作用及诱导同种移植免疫低反应性的机制。方法:分离BALB/c小鼠的骨髓来源树突状细胞,试验分为2组:①脂多糖(LPS)刺激组,即培养结束前加入外源性脂多糖(1 mg/L)刺激24小时;②Crry处理组,加入抗小鼠Crry抗体5μg/ml,结束前加入外源性脂多糖(1 mg/L)刺激24小时,流式细胞仪检测树突状细胞表面分子CD86、CD40、MHCⅡ的表达,ELISA法检测其细胞上清液中IFN-γ、IL-10和IL-12的水平,ELISA法检测细胞上清液中C3、C5、C3a和C5a的含量,并以BALB/c小鼠的DC作为刺激细胞,以C57BL/6小鼠的淋巴细胞作为反应细胞进行同种混合淋巴细胞培养,MTT法检测淋巴细胞增殖活性。结果:经LPS刺激后,培养的DC表面共刺激分子CD40、CD86和MHCⅡ分子的表达显著增加(P<0.05);Crry处理组DC表面共刺激分子CD86和MHCⅡ分子的表达较LPS刺激组显著降低(P<0.05),而CD40的表达无显著性差异;Crry处理组培养上清液中C3和C5的含量无明显变化,但C3a、C5a的含量较LPS刺激组显著减少(P<0.05);培养DC上清液中IFN-γ和IL-12的含量较LPS刺激组显著降低(P<0.05),IL-10的含量较LPS刺激组显著增加(P<0.05);培养的DC加入Crry抗体后,其淋巴细胞增殖活性显著降低(P<0.05)。结论:补体调节蛋白Crry可以对DC具有重要的调控作用,可以影响其共刺激分子的表达、补体的生成以及细胞因子的合成等,从而诱导同种移植免疫低反应性,丰富先天免疫对获得性免疫的调节作用。     

负载滋养层细胞抗原对小鼠树突状细胞表型及功能的影响

目的 研究负载滋养层细胞抗原对小鼠髓源性树突状细胞(DC)分化成熟过程的影响,获得致耐受性DC.方法 体外使用粒细胞巨细胞集落刺激因子(GM-CSF)诱导小鼠骨髓细胞定向分化、经LPS刺激获得成熟DC;通过外胎盘锥组织块培养法获得滋养层细胞,制备可溶性抗原,加入DC培养体系.流式细胞术检测DC表面共刺激分子及MHC-Ⅱ的表达,ELISA法检测DC分泌IL-10和IL-12的浓度,混合淋巴细胞培养评估 DC刺激同种T细胞增殖、活化的功能.结果 成熟DC表型为CD40high CD80highCD86highMHC-Ⅱhigh,分泌大量的IL-12和极少量的IL-10 ,体外能有效刺激T细胞的增殖;负载滋养层细胞抗原的DC表型为CD40midCD80lo wCD86lowMHC-Ⅱlow,在分泌大量IL-12的同时IL-10也明显升高,不能有效刺激T细胞增殖,并使T细胞分泌细胞因子呈现明显Th2偏倚.结论 负载滋养层细胞抗原后的DC表面共刺激分子及MHC-Ⅱ表达降低,刺激T细胞增殖能力下降;其自分泌和促使T细胞旁分泌的细胞因子呈现Th2偏倚,是一种耐受性DC.     

4种细胞因子组合方式对小鼠树突状细胞分化发育的影响

目的观察在体外培养时4种细胞因子(CK)组合方式对小鼠骨髓源树突状细胞(DC)分化、增殖、发育的影响.方法用不同的CK定向诱导小鼠骨髓细胞分化为DC,通过流式细胞仪(荧光抗体双标记法)测定CD11c+细胞比例、MHC-Ⅱ类分子的表达及在脂多糖(LPS)刺激后CD86表达的变化.结果GM-CSF+IL-3+SCF促进DC分化、增殖的能力明显高于其他3组(P＜0.05).该组CK所诱导的DC在LPS刺激后,CD86表达增加的幅度明显低于GM-CSF+IL-4组(P＜0.01).结论GM-CSF、IL-3和SCF对于促进小鼠骨髓细胞向DC定向分化、增殖有协同作用,分化后的DC多数处于发育早期,DC前体所占的比例较大.     

LPS持续刺激对小鼠骨髓树突状细胞成熟的影响

目的　研究LPS持续刺激对小鼠骨髓树突状细胞 (DC)成熟的影响。方法　小鼠骨髓细胞用GM CSF培养 7d ,持续刺激组全程加入LPS ,短期刺激组在最后 48h加入LPS ,对照组不加LPS。流式细胞仪检测细胞表型和细胞摄取抗原的能力 ,ELISA检测细胞产生的细胞因子 ,混合淋巴细胞培养检测细胞提呈抗原的能力。结果　用LPS持续刺激的小鼠骨髓DC表达MHCⅡ、CD86、CD80和CD11c等分子和分泌TNF α和IL 12 (p70 )的能力并未增加 ,吞噬FITC OVA的能力显著升高 ,刺激同种异基因T细胞和刺激同种同基因T细胞增殖的能力亦显著低于LPS短期刺激组。结论　LPS持续刺激可抑制DC的发育成熟 ,可能是持续严重感染时免疫功能低下的原因     

LPS介导的树突状细胞成熟过程中的表型变化

目的:探讨树突状细胞成熟过程中,DC表面MHC分子和共刺激分子的表达变化及MHCⅡ的胞内分布变化。方法:制备小鼠骨髓来源的树突状细胞,LPS分别刺激0、3、6、12和24小时,荧光抗体标记后,用流式细胞仪检测MHCⅠ、MHCⅡ分子和CD86、CD80、CD40等共刺激分子在细胞表面的表达,同时以激光共聚焦显微镜观察MHCⅡ的胞内分布变化。结果:在LPS刺激后,DC细胞表面的不同表型分子,其表达水平随时间延长有不同的上升趋势。同时在未成熟DC中,MHCⅡ主要集中在细胞核附近,LPS刺激后,MHCⅡ朝细胞外围扩散,到刺激12小时,有较多的MHCⅡ出现在细胞表面。结论:LPS介导的树突状细胞成熟过程中的表型分子有不同的变化趋势。     

TLR2激动剂Pam3CK逆转IL-10转染小鼠髓样树突状细胞免疫功能的研究

目的:观察TLR2激活剂Pam3CK对IL-10转染小鼠髓样树突状细胞免疫功能影响。方法:转染IL-10至小鼠髓样树突状细胞(mDC),TLR2配体Pam3CK刺激48小时,利用流式细胞仪检测DC表面标志MHCⅡ、CD80、CD86及FasL等分子的表达;ELISA检测细胞产生IL-6、TNF-α。结果:IL-10抑制mDC表达CD80、CD86、MHCⅡ类分子,降低其分泌IL-6、TNF-α,促进其表达FasL,而TLR2激动剂刺激增加了IL-10转染DC表达MHC-Ⅱ类分子及CD80、CD86,促进其产生IL-6及TNF-α,抑制了FasL表达。结论:TLR2激动剂可逆转IL-10诱发的DC免疫应答低下。     

microRNA-27a对树突状细胞表型及功能的影响

目的:研究microRNA-27a(miR-27a)对脂多糖(Lipopolysaccharide,LPS)刺激的小鼠树突状细胞(Dendritic cell,DC)的成熟和细胞因子分泌的影响。方法:小鼠骨髓来源的未成熟树突状细胞(immature dendritic cell,im DC)转染miR-27a的模拟物(miR-27a mimics)后,用LPS刺激24 h,采用流式细胞仪检测其表面共刺激分子CD80、CD86及MHCⅡ表达,ELISA方法检测其上清中的IL-12p70及IL-10蛋白水平,RT-PCR方法检测其细胞内IL-12p40及IL-10 mRNA水平,混合淋巴细胞反应(MLR)检测其刺激T细胞增殖能力。结果:与未处理的im DC比较,LPS刺激24 h后的DC表面的共刺激分子CD80、CD86及MHCⅡ表达均显著增高(均P0.001);LPS刺激24 h后,与对照组比较,转染miR-27a mimics细胞的共刺激分子CD80、CD86及MHCⅡ表达均显著降低(均P0.001),且显著抑制IL-12分泌(P0.01)、促进IL-10分泌(P0.05),并显著减弱LPS刺激的DC促CD4+T细胞增殖的能力(P0.01)。结论:miR-27a影响小鼠树突状细胞的成熟以及细胞因子的分泌。     

Dexamethasone induces IL-10-producing monocyte-derived dendritic cells with durable immaturity

It is highly desirable that immature dendritic cells (DC) used for tolerance induction maintain steady immature state with predominant interleukin (IL)-10 production. In this study, we attempted to develop DC with durable immaturity and other tolerogenic features by using dexamethasone (Dex). We found DC derived from human monocytes in the presence of 10(-7) m Dex were negative for CD1a. Compared with control transduced DC (Ctrl-DC), Dex-DC expressed lower CD40, CD80 and CD86 but equivalent human leucocyte antigen-DR. Both immature Dex- and Ctrl-DC did not express CD83. Nevertheless, upon stimulation of lipopolysaccharide (LPS) or CD40 ligand, the expression of CD40, CD80, CD83 and CD86 was upregulated on Ctrl-DC but not on Dex-DC. The immaturity of Dex-DC was durable following Dex removal. Interestingly, Dex-DC maintained production of large amount of IL-10 and little IL-12 five days after Dex removed. Further study indicated that high-level IL-10 production by Dex-DC was associated with high-level phosphorylation of extracellular signal-regulated kinase (ERK) as blockade of this enzyme markedly attenuated IL-10 production. Furthermore, Dex-DC sustained the capability of high phosphorylation of ERK and IL-10 production 5 days after Dex removal. In addition, Dex-DC had significantly lower activity in stimulating T-cell proliferation. Neutralization of IL-10, to some extent, promoted DC maturation activated by LPS, as well as T-cell stimulatory activity of Dex-DC. The above findings suggest that IL-10-producing Dex-DC with durable immaturity are potentially useful for induction of immune tolerance.     

The glucocorticoid dexamethasone programs human dendritic cells for enhanced phagocytosis of apoptotic neutrophils and inflammatory response

GCs are powerful anti-inflammatory compounds inhibiting inflammatory cell recruitment and production of proinflammatory cytokines. We have recently found that DCs, the key players of T cell priming and polarization, respond to allogeneic apoptotic neutrophils with proinflammatory cytokine release and Th1 cell activation. Here, we show that monocyte-derived human DCs develop their capacity to engulf apoptotic cells by up-regulating a set of apoptophagocytic genes. This gene expression pattern was reprogrammed when differentiation took place in the presence of the synthetic GC Dex, which increased the expression of phagocytosis receptors MERTK and CD14, the bridging molecule C1QA, DNASE2, and ADORA3. The increased phagocytosis was attenuated by the addition of ADORA3 antagonist and could not be observed when bone marrow-derived DCs of ADORA3 KO mice were treated with Dex. The GC-treated human DCs loaded with allogeneic apoptotic neutrophils secreted, in response to LPS and IFN-γ, the inflammatory cytokine TNF-α. Furthermore, the Dex-treated DCs could activate autologous T lymphocytes toward Th1 effector cells, and this was enhanced by their exposure to allogeneic apoptotic neutrophils.     

核转录因子RelB抑制途径对骨髓树突状细胞表面分子表达的影响研究

目的探讨核转录因子RelB抑制途径对小鼠骨髓树突状细胞（bone marrow dendritic cells）的表面分子表达的影响,为致耐受树突状细胞的研究提供新方法。方法 rmGM-CSF和rmIL-4联合诱导培养体系培养小鼠骨髓树突状细胞,免疫磁珠方法纯化;用慢病毒载体制备RelB shRNA慢病毒,与小鼠骨髓树突状细胞共培养,流式细胞术观察树突状细胞表面分子MHC-II、CD86和CD40的表达,设LPS-DC对照组、未处理组和LPSRNAi RelB DC组。结果核转录因子RelB抑制的树突状细胞表面分子MHC-II、CD86和CD40均低水平表达,显著低于成熟DC表面分子的表达（P〈0.05）,且经LPS刺激后（LPS RNAi RelB DC）DC表面上述三类分子的表达水平仍显著低于LPS-DC组（P〈0.05）,与未处理组（immature DC）表面分子表达水平相当。结论核转录因子RelB抑制的骨髓树突状细胞表面分子表达水平低,呈现出致耐受的树突状细胞的特点,是致耐受树突状细胞研究的一种新方法。     

IL-17A 协同GM-CSF 和LPS 促进骨髓细胞衍生树突状细胞的分化和成熟研究

目的:探讨IL鄄17A 对小鼠骨髓细胞衍生树突状细胞分化和成熟的影响。方法:分离小鼠骨髓细胞,加入含GM-CSF(20 ng/ ml)RPMI1640 完全培基培养8 d,诱导小鼠骨髓单个核细胞向DC 分化,加入LPS(1 滋g/ ml)继续培养36 h,进一步诱导DC 成熟,同时在骨髓细胞衍生诱导DC 分化及成熟的不同阶段加入不同浓度的rmIL-17A(10、100 ng/ ml),采用流式细胞术检测DC 表面共刺激分子的表达,ELISA 方法检测DC 培养上清中IL-12p40 和IL-10 水平。结果:rmIL-17A 可促进GM-CSF 诱导骨髓细胞衍生DC 表面共刺激分子CD40、CD80、CD86 和MHC域的表达,且具有剂量依赖性,其中以高浓度rmIL-17A刺激组的CD40 及MHC域表达增加最显著;在LPS 诱导DC 成熟阶段加入rmIL-17A,骨髓细胞衍生DC 共刺激分子CD40、CD80、CD86 和MHC域的表达均明显增加,并且随着rmIL-17A 浓度的增加,CD86 和MHC域的表达水平也随之增高;同时与未加rmIL鄄17A 的对照组相比,低浓度rmIL-17A 组LPS 刺激骨髓细胞衍生DC 分泌IL-12p40 和IL鄄10 水平均显著增加(P <0.001),高浓度rmIL-17A 组IL-12p40 水平显著增高(P<0.001),但IL-10 水平没有变化。结论:IL-17A 可促进GM-CSF 诱导的骨髓细胞衍生DC 前体细胞表型发展,并能协同LPS 诱导骨髓衍生DC 的分化和成熟。     

Dexamethasone inhibits the antigen presentation of dendritic cells in MHC class II pathway

Glucocorticoids (GC) are physiological inhibitors of inflammatory responses and are widely used as anti-inflammatory and immunosuppressive agents in treatment of many autoimmune and allergic diseases. In the present study, we demonstrated that one of the mechanisms by which GC can suppress the immune responses is to inhibit the differentiation and antigen presentation of dendritic cells (DC). DC were differentiated from murine bone marrow hematopoietic progenitor cells by culture with GM-CSF and IL-4 with or without dexamethasone (Dex). Our data showed that Dex, in a dose dependent manner, down-regulated surface expression of CD86, CD40, CD54 and MHC class II molecules by DC, but the expression of MHC class I, CD80, CD95 and CD95L were not affected. In addition, Dex-treated DC showed an impaired function to activate alloreactive T cells and to secrete IL-Ibeta and IL-12p70. Moreover, Dex inhibited DC to present antigen by MHC class II pathway. However, the endocytotic activity of DC was not affected. The inhibitory effect of Dex on the expression of costimulatory molecules and the antigen-presenting capacity of DC could be blocked by the addition of RU486, a potent steroid hormone antagonist, suggesting the requirement of binding to cytosolic receptors in the above-described action of Dex. Since DC have the unique property to present antigen to responding naive T cells and are required in the induction of a primary response, the functional suppression of DC by Dex may be one of the mechanisms by which GC regulate immune responses in vivo.     

Generation and characterization of an immunogenic dendritic cell population

Dendritic cells (DCs) capture, internalize and process antigens leading to the induction of antigen-specific immune responses. The aim of this study was to develop, implement and characterize an efficient approach for DC-based immunization. Dendritic cells were expanded in vivo by hydrodynamic delivery of a human flt3 ligand expression plasmid. Splenic DCs were isolated and purified with magnetic beads linked to hepatitis C virus (HCV) nonstructural protein-5 (NS5), anti-CD40 and/or LPS. The DCs that contained beads were purified by passage over a magnetic column and subsequently phenotyped. Enrichment resulted in a population consisting of 80% CD11c(+) cells. Uptake of uncoated microparticles promoted DC maturation and the expression of CD80, CD86, and MHC-II molecules; beads coated with LPS and anti-CD40 further increased the expression of these co-stimulatory molecules, as well as the secretion of IL-12. Mice immunized subcutaneously with DCs containing beads coated with HCV NS5 protein, anti-CD40 and LPS exhibited significant antigen-specific, increases in IFN-gamma-producing CD4(+) T cells and CTL activity. This approach combines three critical elements necessary for efficient DC-based immunization that include DC enrichment, maturation and antigen targeting.     

Triptolide inhibits IL-12 production and T cell-stimulatory capacity of dendritic cells

Extracts of Tripterygium wilfordii Hook F.(TWHF ) are effective in traditional Chinesemedicine for treatment of autoimmune diseasessuch as rheumatoid arthritis, systemic lupus ery thematosus, nephritis and asthma [1, 2]. Trip tolide, a diterpenoid triepoxide, is derived fromTWHF and is responsible for most of the immuno suppressive and anti inflammatory effects ofTWHF. Triptolide has been shown to be effectivein the treatment of autoimmune diseases, …     

Activation of purified allogeneic CD4(+) T cells by rat bone marrow-derived dendritic cells induces concurrent secretion of IFN-gamma, IL-4, and IL-10

Dendritic cells (DCs) are highly specialized antigen-presenting cells that play a key role in the initiation and regulation of immune responses. The ability of DCs to process antigens and the outcome of their interaction with T cells are largely dependent on phenotype as well as maturation state of DCs. In this study, we generated DCs from rat bone marrow precursors. Bone marrow cells cultured in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4, and Flt-3 ligand (FL) produced immature DCs that expressed intermediate levels of major histocompatibility complex (MHC) class II, low levels of CD80 and CD86 molecules and displayed a high capacity of endocytosis. Bone marrow-derived DCs (BMDCs) matured in the presence of lipopolysaccharide (LPS) upregulated expression of MHC class II, CD80 and CD86, while their phagocytic capacity was dramatically reduced. Mature BMDCs stimulated vigorous proliferation of purified allogeneic CD4(+) T cells in a primary mixed leukocyte reaction (MLR) and elicited a mixed cytokine profile in allogeneic CD4(+) T cells: DCs activated CD4(+) T cells to produce interferon (IFN)-gamma, IL-4, and IL-10. Thus, rat BMDCs effectively internalize antigens and stimulate T cell proliferation but fail to induce an unidirectional polarization of T helper (T(H)) cells and in this respect differ from both human and mouse DCs.