RelB shRNA慢病毒感染小鼠骨髓树突状细胞的免疫学效应

背景：沉默树突状细胞发育成熟的关键基因核因子кB/RelB可构建新型致耐受树突状细胞？ 目的：探讨RelB shRNA转染小鼠骨髓树突细胞的生物免疫学功能的影响。 方法：利用重组粒细胞-巨噬细胞集落刺激因子和重组白细胞介素4联合诱导小鼠骨髓树突状细胞;慢病毒载体将RelB shRNA转染致小鼠骨髓树突细胞后,分为未成熟树突状细胞、脂多糖刺激成熟、RelB基因沉默及脂多糖刺激RelB沉默的4组树突状细胞进行观察。 结果：体外培养第 6 天脂多糖刺激组细胞表面可见大量细长的类似树枝的突起,其他3组细胞形态特征相似,呈圆形、皱缩状态,这3组细胞表面MHC-II类分子、CD86和CD40分子表达水平相当,但低于脂多糖刺激组;3组混合淋巴细胞反应中刺激T细胞增殖能力差异无显著性意义(P > 0.05),但均较脂多糖刺激组显著降低(P < 0.01);RelB基因沉默的树突状细胞分泌Th1细胞因子γ-干扰素和白细胞介素2的能力较低,分泌Th2白细胞介素10和白细胞介素4的能力较高(P < 0.01),Th1/Th2细胞因子的比例与未成熟树突状细胞类似。说明RelB shRNA经慢病毒转染骨髓源性树突状细胞后,在细胞形态、表面分子表达、免疫学功能等方面均具有与未成熟树突状细胞相似的特点,且不能被脂多糖刺激成熟。     

RNAiRelBDC对CD4＋CD25＋双阳性T细胞诱导效应的实验研究

目的:探讨核转录因子NF-кB/RelB基凶沉默的树突状细胞(RNAi RelB dendritic cell,RNAi helB DC)诱导异基因CD4+CD25+双阳性T细胞牛成的生物效应.方法:实验分为Control-DC组、LPS-DC组、RNAi RelB DC组和LPS-RNAiRelB DC组,各组DC均与同种异基因T淋巴细胞混合反应后,采用流式细胞术(FCM)分析各组CD4+CD25+双阳性T细胞牛成比例.结果:Control-Dc组DC较LPS-Dc组DC诱导CD4+CD25+双阳性T细胞的增高幅度更显著,两者差异具有统计学意义(P＜0.01).RNAi RelB DC组和LPS-RNAiRelB DC组DC诱导CD4+CD25+双阳性T细胞牛成比例与LPS-DC组相比均存在显著差异(P＜0.01),RNAi RelB DC组DC诱导CD4+CD25+双阳性T细胞牛成能力较Control-DC组强,差异具有统计学意义(P＜0.05).结论:RelB基凶被沉默后DC具有较强的诱导CD4+CD25+双阳性T细胞生成的能力,表现出未成熟DC的特点,这种RNAi RelB DC有望成为一种新型的致耐受DC用于免疫耐受的诱导研究.     

慢病毒载体介导的小鼠骨髓树突状细胞RelB基因沉默

目的: 利用慢病毒载体沉默小鼠骨髓树突状细胞(DC)的RelB基因, 建立RelB基因低表达的骨髓致耐受DC, 为自身免疫病的防治提供新方法.方法: 设计小鼠RelB基因干扰的靶序列R5, 合成并克隆到慢病毒载体上, 与慢病毒的包装材料在293FT细胞中包装成病毒颗粒, 收集病毒上清, 测定病毒滴度, 然后包装慢病毒感染DC, RT-PCR和免疫荧光方法检测, 灰度扫描并用软件分析RelB基因的表达水平, 未成熟DC作对照组, 选择T6包装病毒为RNAi的阴性对照.结果: RT-PCR和免疫荧光方法发现R5病毒感染的DC RelB基因表达水平与未成熟DC基因表达水平接近, 低于成熟DC的表达水平(P<0.05), 而实验对照T6组病毒感染的DC其RelB基因表达水平高于未成熟DC(P<0.05)和R5病毒转染DC组(P<0.05).结论: 小鼠骨髓DC表达的RelB基因被慢病毒载体有效沉默, 有望成为DC免疫治疗的新载体.     

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。     

小鼠骨髓成熟与不成熟树突状细胞中RelB基因的表达

目的:探讨体外分离培养的小鼠骨髓来源的成熟与未成熟DC中核转录因子RelB(avianreticuloendotheliosisviral(v-rel)oncogenerelatedB)基因的表达。方法:无菌从C57BL/6小鼠股骨和胫骨中取出骨髓细胞,利用rmGM-CSF和rmIL-4联合诱导骨髓前体细胞产生未成熟的DC,未成熟的DC在培养结束前18h经LPS刺激获得成熟的DC,用流式细胞术分析它们的表型,用RT-PCR和免疫荧光染色法检测成熟与未成熟DC中,RelBmRNA和其蛋白的表达。结果:流式细胞术分析显示未成熟的DC中MHC-Ⅱ类分子和共刺激分子(CD86和CD40)呈低水平表达;而成熟的DC则呈高水平表达。RT-PCR和免疫荧光染色法检测结果均显示,RelB基因在未成熟的DC中呈低水平表达;而在成熟的DC中呈高水平表达,两者比较具有统计学意义(P<0.01)。结论:RelB基因的表达与小鼠骨髓来源的DC的成熟状态密切相关。抑制DC中RelB基因的表达,有可能诱导产生具有耐受原性的未成熟的DC。     

IL-10诱导小鼠树突状细胞耐受的分子机制

目的：研究白细胞介素-10 (interleukin-10，IL-10)诱导小鼠来源的树突状细胞(DC)耐受及其与配对免疫球蛋白样受体（PIR-A/B）的关系。方法: 以IL-10（20 μg/L）诱导小鼠来源的树突状细胞系（DC2.4）6 d，即IL-10-DC组，脂多糖（LPS）刺激其48 h为成熟DC2.4细胞（LPS-DC），体外化学合成特异性针对PIR-B的小干扰RNA片段，以脂质体2 000转染IL-10组（Si-DC组）。分别应用半定量RT-PCR和流式细胞仪（FCM）检测DC2.4、IL-10组、LPS组及Si-DC组细胞PIR-A/B的表达。以［3H］-TdR标记法检测上述各组细胞刺激同种异体淋巴细胞的增殖反应(MLR)，ELISA方法测混合培养上清中IFN-γ的水平变化。结果: RT- PCR结果表明，IL-10诱导PIR-B表达升高、PIR-A表达下降，LPS则下调PIR-B、上调PIR-A的表达。FCM检测IL-10组和LPS组的PIR-A/B胞外区PIR表达均升高，且前者明显高于后者。同正常DC2.4和LPS组相比，IL-10可抑制MLR，小干扰RNA沉默PIR-B表达可增强MLR，伴随MLR反应上清中IFN-γ的水平升高。结论: IL-10诱导DC高度表达免疫抑制性受体PIR-B，使其获得耐受，上调PIR-B的表达是IL-10诱导DC耐受的分子机制之一。     

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。     

雷帕霉素和地塞米松对小鼠树突状细胞分化成熟的调控

目的：观察雷帕霉素（rapamycin，Rap）和地塞米松（dexamethasone，Dex）对堵养的小鼠骨髓来源的树突状细胞（DC）分化发育的影响。方法：（1）用GM-CSF＋IL-4定向诱导C57BL／6小鼠骨髓细胞分化为DC，分别加入Rap或Dex，然后用脂多糖（LPS）刺激。在倒置显微镜和扫描电镜下，动态观察DC形态学E的变化。（2）通过流式细胞术（荧光抗体双标记法）测定CD11c^＋细胞的比例及CD86和MHC-Ⅱ类分子表达的变化。（3）通过单向混合淋巴细胞反应（MLR）观察，Rap和Dex处理的DC刺激BALB／c小鼠同种异基因T细胞增殖的情况。结果：（1）经Rap和Dex处理后，DC在形态学上呈现稳定不成熟状态。（2）Rap处理的细胞表面CDIlc和MHC-Ⅱ类分子的表达仅有轻度降低，而CD86的表达明显降低。Dex处理的细胞表面CDIlc的表达与Dex的剂量呈负相关，CD86和MHC-Ⅱ类分子的表达均明硅降低。两种药物处理的DC均可抵抗LPS的促成熟作用。（3）MLR的结果显示，Rap和Dex处理的DC刺激同种异基因BALB／c小鼠T细胞增殖的能力均较低。结论：Rap和Dex均可使DC处于稳定的不成熟状态。与Dex相比，Rap对骨髓造血F细胞向DC分化的过程影响较小，而且在抑制DC表面协同刺激分子CD86表达的同时，对MHC-Ⅱ类分子的表达影响较小。     

缺氧和脂多糖促进小鼠树突状细胞自噬并增强表面共刺激分子CD40、 CD80的表达

目的研究缺氧和脂多糖(LPS)诱导的树突状细胞(DC)自噬发生及表面共刺激分子CD40、 CD80的表达情况。方法采用小鼠DC,分为正常组、缺氧组、脂多糖(LPS)处理组、缺氧联合LPS处理组。各组分别处理24 h,噻唑蓝(MTT)法检测细胞增殖水平变化;Western blot法检测自噬微管相关蛋白1轻链3Ⅰ(LC3Ⅰ)、LC3Ⅱ和beclin1的蛋白水平;透射电镜观察LPS处理后DC自噬小体的形成;流式细胞术检测DC表面CD40、 CD80的变化;免疫荧光细胞化学染色检测小鼠DC表面CD40、 CD80的表达。结果缺氧及LPS均能诱导小鼠DC发生自噬,增加其表面CD40、 CD80的表达。缺氧联合LPS处理后,小鼠DC自噬发生明显加强,自噬小体增多,其表面共刺激分子CD40、 CD80的表达增多。结论缺氧及LPS均能引起小鼠DC发生自噬并增加CD40、 CD80的表达。     

IL-37抑制脂多糖诱导的小鼠树突状细胞活化

目的探讨白细胞介素37(IL-37)对细菌脂多糖(LPS)诱导的小鼠树突状细胞(DC)活化的调节作用。方法应用GM-CSF和IL-4诱导小鼠骨髓细胞向DC分化,抗CD11c磁珠分选DC。IL-37预处理DC后,进行LPS刺激。流式细胞术检测DC表面共刺激分子(CD80、CD86)表达水平,实时荧光定量PCR检测肿瘤坏死因子α(TNF-α)、IL-6和IL-1αmRNA表达水平,流式细胞微球芯片试剂盒(CBA试剂盒)检测细胞培养上清中IL-1α、IL-6、TNF-α等因子的浓度。结果 DC诱导成功,磁珠分选能够获得高纯度的DC(>90%)。IL-37降低LPS诱导的DC表面共刺激分子CD80、CD86的表达,并抑制DC合成IL-1α、IL-6、TNF-α。结论 IL-37可以通过降低共刺激分子和炎症因子的表达抑制LPS刺激的DC活化。     

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。     

Impaired dendritic cell differentiation and maturation in the absence of C3

Human monocytes can be differentiated into immature dendritic cells (DCs) in the presence of serum and cytokines. One of the main functions of immature DCs is to capture and process antigens. Following maturation, they differentiate into antigen presenting cells. The role of complement in the differentiation process from monocytes towards immature DCs remains elusive. Here we demonstrate that complement 3 (C3) has a regulatory impact on the expression of specific DC surface molecules and DC-derived cytokine production during DC differentiation. We isolated human adherent peripheral blood mononuclear cells, which were cultured in the presence of GM-CSF plus IL-4 in medium supplemented with normal human serum or C3 deficient serum. The lack of C3 during DC differentiation negatively impacted the expression of C-type lectin receptor DC-SIGN, the antigen presenting molecules HLA-DR and CD1a, and the costimulatory molecules CD80 and CD86. Further, the spontaneous production of IL-6 and IL-12 was reduced in the absence of C3. Moreover, the maturation of immature DCs in response to LPS challenge was impaired in the absence of C3 as evidenced by reduced MHC-II, co-stimulatory molecule expression as well as modulated IL-12 and TNF-alpha production. Collectively, our results provide evidence for a novel role of C3 as a critical cofactor in human DC differentiation and maturation.     

重组大鼠4-1BBL对树突状细胞免疫学功能的影响

目的:探讨大鼠4-1BBL对骨髓来源的树突状细胞(DC)表面分子及免疫功能的影响.方法:构建真核表达质粒pIRES2-EGFP- 4-1BBL并利用脂质体介导转染HepG2细胞, G418筛选出阳性克隆, RT-PCR和荧光显微镜分别检测4-1BBL基因转录和EGFP的表达; 流式细胞术(FCM)检测不同HepG2细胞与DC共培养2 d后表面CD80及MHC-Ⅱ的表达, ELISA测定共培养上清中IL-6和IL-12的含量.结果:成功构建pIRES2-EGFP- 4-1BBL重组表达载体并在HepG2细胞中获得稳定表达, 重组大鼠4-1BBL能促进骨髓来源DC表面MHC-Ⅱ、 CD80分子表达和分泌IL-6、 IL-12(P<0.05).结论:重组大鼠4-1BBL具有促进骨髓来源DC成熟的功能.     

Differential up-regulation of HLA-DM, invariant chain, and CD83 on myeloid and plasmacytoid dendritic cells from peripheral blood

Two main dendritic cell (DC) subsets have been described in peripheral blood, the myeloid subset or DC1 that is characterized by the presence of CD11c and the plasmacytoid subset or DC2 negative for this marker. The two subsets may perform different functions and have been defined as immunogenic (the myeloid subset) or tolerogenic (the plasmacytoid subset). The expression of human leukocyte antigen (HLA)-DM molecules, which act as peptide editors in the antigen presentation process, was studied in freshly isolated plasmacytoid and myeloid DCs from peripheral blood. The expression of the invariant chain (Ii), the major histocompatibility complex class II (MHC-II) : class II-associated Ii peptide (CLIP) complex, and CD83 was also investigated. The results showed that intracellular expression of HLA-DM and the Ii was significantly higher in the plasmacytoid than in the myeloid DC subset. In contrast, a higher fraction of cell expressing MHC-II : CLIP complex was found in the myeloid than in the plasmacytoid DC subpopulation. CD83 was not detected in any of these two subsets. Following culture of these cells with interleukin-3 (IL-3), tumor necrosis factor-alpha (TNFalpha) and/or heat shock protein-70 (HSP-70), the expression of intracellular HLA-DM was up-regulated in the myeloid DCs to levels similar to those found in the plasmacytoid DCs, whilst the Ii was down-regulated in the plasmacytoid subset to similar levels to those expressed in the myeloid DCs. In addition, CD83 was up-regulated in the myeloid (CD11c+) but not in the plasmacytoid (CD11c-) DCs. The expression pattern of these antigen-processing molecules could be related to the immaturity and function attributed to these DC subsets.     

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.