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.     

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。     

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。     

慢病毒载体介导的IL-10诱导耐受性树突状细胞的形成

探讨通过IL-10重组慢病毒载体转染小鼠未成熟树突状细胞(iDC),构建高分泌IL-10的耐受性树突状细胞的可行性.粒细胞巨噬细胞集落刺激因子(GM-CSF)和白细胞介素4(IL-4)联合诱导并经CD11c免疫磁珠分选获得小鼠骨髓iDC,倒置显微镜下观察,流式细胞术分析细胞表面分子:IL-10重组慢病毒载体转染小鼠iD...     

罗勒多糖对树突状细胞表面分子表达的影响

目的：观察罗勒多糖对人外周血单核细胞来源的树突状细胞（Dendritic cell，DC）表面分子表达的影响，探讨其抗肿瘤免疫机制。方法：从正常人外周血分离获得单核细胞，加入含10％胎牛血清、CM-CSF及IL-4的RPMI1640，37℃培养5天，实验组加入罗勒多糖，对照组加入PBS，流式细胞仪检测细胞表面分子的表达。结果：在细胞因子的诱导下，CD14^＋单核细胞逐渐分化为DC，罗勒多糖作用组与对照组DC均表达CD209、CD80、CD83、CD86、CD1a和HLA-DR，与对照组相比，罗勒多糖组DC表面分子CD80和HLA-DR的表达均明显上调。结论：罗勒多糖能够调节DC表面分子CD80和HLA-DR的表达，这可能是罗勒多糖发挥其抗肿瘤免疫的机制之一。     

FasL基因重组慢病毒载体感染SD大鼠树突状细胞的研究

目的 探讨FasL基因重组慢病毒载体感染SD大鼠树突状细胞的效率和FasI 蛋白的表达情况,为进一步研究转FasL基因在同种异体器官移植中诱导免疫耐受和保护移植物打下基础.方法 将培养一周的细胞重铺于六孔板中,每孔细胞数量为5×105,24 h后观察,细胞适合感染,按照MOI=10感染细胞,使用GFP阳性对照质粒作对照实验,感染24 h后,培养皿中添加1 ml新鲜培养基,每隔1 d加细胞因子继续培养,荧光显微镜观察荧光强度和数量,添加病毒液后10 d收集细胞进行实时定量检测和WB检测.结果 FasL基困重组慢病毒载体感染DC 8 d后,细胞开始出现荧光,10 d感染效率为100%;实时定量PCR检测瞬时转染后目的 基因的表达显示以细胞的1.00%为参照,Cell+FasL质粒为167.03%;免疫印迹检测转染后FasL蛋白的表达显示以细胞的1.00%为参照,细胞+FasL质粒为34.15%.结论 FasL基因重组慢病毒载体成功感染DC,实时定量PCR及Western印迹证实感染的Dc表达FasL明显提高.为进一步研究转FasL摹因在同种异体器官移植中诱导免疫耐受和保护移植物打下基础.     

耐受性树突状细胞抑制炎症性疾病的机制及进展

树突状细胞(DCs)是调节免疫反应的关键细胞,通过呈递抗原参与机体的固有免疫和适应性免疫。耐受型DC(tolDC)主要通过抑制效应T细胞反应及诱导调节性T细胞(Tregs)发挥免疫耐受作用。tolDC以致免疫耐受这一特点被广泛应用于自身免疫病和器官移植的治疗,其是否可用于炎症、过敏性疾病的治疗已成为近年的一大研究热点。本文对tolDC的细胞分型、表面标志物,其参与抑制炎症反应可能的免疫细胞、酶、细胞因子、信号转导通路等机制进行总结,并对其在炎症相关疾病的基础和临床研究应用作一综述。     

未成熟树突状细胞对T细胞活化及其IFN-γ和IL-12表达的影响

目的:探讨未成熟树突状细胞(DC)对T细胞活化及其IFN-γ和IL-12表达的影响。方法:通过诱导Lewis大鼠骨髓前体细胞,获取未成熟(iDC)和成熟(mDC)两种状态的DC;将两种DC用乙酰胆碱受体(AChR)负载后进行T细胞重复刺激、交叉刺激和无关抗原刺激试验,观察T细胞增殖情况并测定T细胞IFN-γ、IL-12的表达水平。结果:(1)AChR负载的iDC可明显抑制T细胞增殖,且这种被抑制的T细胞对AChR负载的mDC的交叉刺激也不引起增殖,但对无关抗原OVA负载的mDC的刺激可产生明显增殖。(2)与mDC组比较,AChR负载的iDC初次和重复刺激均明显抑制T细胞IFN-γ和IL-12的表达。结论:iDC可诱导抗原特异性T细胞耐受,耐受机制可能与Th1细胞因子IFN-γ和IL-12的抑制有关。     

预输注RelB shRNA慢病毒修饰树突细胞诱导大鼠肝移植免疫耐受

背景：肝移植后的排斥反应是威胁患者和移植物长期存活的主要原因。诱导受者产生特异性免疫耐受是解决排斥反应的理想措施。 目的：探讨RNAi RelB树突状细胞预输注诱导大鼠肝移植特异性免疫耐受的可能性。 方法：将近交系雄性清洁级DA(RT1a)大鼠和近交系雄性SPF级Lewis(RT11)大鼠分别作为供、受体,行原位肝移植手术。术前随机配对分为4组：①对照组,受体鼠移植前不做预输注。②治疗组：受体鼠移植前7 d静脉输注供体大鼠RNAi RelB树突状细胞(5×106)。③未成熟树突状细胞组：受体鼠移植前7 d静脉输注供体大鼠未成熟树突状细胞(5×106)。④成熟树突状细胞组：受体鼠移植前7 d静脉输注供体大鼠成熟树突状细胞(5×106)。 结果与结论：与对照组、未成熟树突状细胞组以及成熟树突状细胞组相比,治疗组移植肝的平均生存时间显著延长。移植后第7天,治疗组天冬氨酸转氨酶,总胆红素水平低于对照组、成熟树突状细胞组、未成熟树突状细胞组(P < 0.01),移植后第14天治疗组、未成熟树突状细胞组天冬氨酸转氨酶,总胆红素均有轻微下降,两组比较差异仍有显著性意义(P < 0.01)。移植后第7天,与治疗组比较,对照组、成熟树突状细胞组、未成熟树突状细胞组γ-干扰素、白细胞介素2水平升高(P < 0.01),而白细胞介素4、白细胞介素10下降(P < 0.01);移植后第14天治疗组、未成熟树突状细胞组γ-干扰素、白细胞介素2水平均有下降,白细胞介素4、白细胞介素10水平均有升高,两组比较差异仍有显著性意义(P < 0.01)。对照组、成熟树突状细胞组、未成熟树突状细胞组移植后第7天排斥活动指数为8.0-9.0。未成熟树突状细胞组第14天肝细胞、内皮细胞坏死及汇管区炎性细胞浸润进一步增多。治疗组移植后第7天排斥活动指数为6.0-8.0,第14天时排斥活动指数为4.0-5.0。结果提示RNAi RelB树突状细胞预输注可以减轻移植肝排斥程度,延长移植肝生存时间,这是通过间接途径实现的,其机制可能与T细胞的调节和无能有关。     

CD40 RNA干扰小鼠树突状细胞抑制异系T淋巴细胞增殖的研究

目的 构建针对小鼠CD40基因的RNA干扰(RNA interference,RNAi)慢病毒载体,制备低表达CD40的树突状细胞(dendritic cells,DC),探讨其阻断CD40/CD40L共刺激通路诱导异系T淋巴细胞无反应的作用机制.方法 采用培养基选择法体外培养小鼠骨髓源DC,构建针对小鼠CD40 RNAi慢病毒载体,体外感染小鼠骨髓源DC.荧光实时定量PCR及流式细胞术检测感染前后CD40 mRNA及DC表面抗原CD40表达.混合淋巴细胞培养观察CD40 RNAi慢病毒感染DC对异系T淋巴细胞增殖能力的影响.结果 体外培养可获得成熟的小鼠骨髓源DC.成功构建小鼠CD40RNAi慢病毒载体,检测滴度为8×10~9 TU/ml.慢病毒感染DC后与感染前相比,明显抑制CD40mRNA的表达(P<0.05),CD40蛋白表达也明显减少(P<0.05).混合淋巴细胞培养结果显示,CD40RNAi慢病毒感染DC组(1.381±0.442)与未感染组(2.444±0.485)及空慢病毒感染DC组(2.294±0.367)相比,淋巴细胞刺激指数(SI)明显下降(P<0.01).结论 培养基选择法可收获大量骨髓源DC,所获骨髓源DC能激活初始型T细胞免疫应答.CD40 RNAi慢病毒感染小鼠骨髓源DC,可以使DC低表达CD40蛋白.CD40 RNAi慢病毒感染的DC体外刺激异系T淋巴细胞增殖的能力下降.为研究CD40 RNAi在同种器官移植诱导免疫耐受奠定基础.     

RelB在蛋白酶体抑制剂诱导的maspin表达中的作用研究

目的: 探讨前列腺癌细胞中,核转录因子NF-κB家族成员之一RelB在蛋白酶体抑制剂作用下maspin蛋白表达调控中的作用机制。方法: 采用Western blotting法检测前列腺癌细胞中内源性及蛋白酶体抑制剂MG-132作用下RelA、RelB和maspin的蛋白表达;采用免疫组化法检测前列腺癌组织中RelB和maspin的表达情况;通过转染小分子RNA至前列腺癌细胞中沉默RelB的表达;采用Western blotting法检测RelB沉默对MG-132诱导的maspin蛋白表达情况的影响;PI染色法结合流式细胞术检测细胞死亡率。结果: 雄激素非依赖型前列腺癌细胞DU145中RelB表达增强而maspin表达明显降低。在检测的10例前列腺癌组织样本中,恶性度高(Gleason评分4-5)的样本普遍同时存在胞核内RelB强染色和maspin表达缺失。MG-132作用DU145细胞24 h后RelB在胞浆和胞核中表达水平下调,伴随着maspin在胞核内的表达上调。MG-132处理RelB表达沉默的DU145细胞8和24 h后,maspin的表达无明显变化,而RelA蛋白的表达水平呈时间依赖性的下调。结论: 在雄激素非依赖型前列腺癌细胞和晚期前列腺癌组织中,maspin与RelB的表达呈负相关性。蛋白酶体抑制剂诱导maspin的表达上调过程中,RelB是重要的调控因子。     

Dendritic cell control of tolerogenic responses

One of the most fundamental problems in immunology is the seemingly schizophrenic ability of the immune system to launch robust immunity against pathogens, while acquiring and maintaining a state of tolerance to the body's own tissues and the trillions of commensal microorganisms and food antigens that confront it every day. A fundamental role for the innate immune system, particularly dendritic cells (DCs), in orchestrating immunological tolerance has been appreciated, but emerging studies have highlighted the nature of the innate receptors and the signaling pathways that program DCs to a tolerogenic state. Furthermore, several studies have emphasized the major role played by cellular interactions and the microenvironment in programming tolerogenic DCs. Here, we review these studies and suggest that the innate control of tolerogenic responses can be viewed as different hierarchies of organization, in which DCs, their innate receptors and signaling networks, and their interactions with other cells and local microenvironments represent different levels of the hierarchy.     

脐血来源的树突状细胞增强CIK细胞的杀伤作用

为确认体外诱导出的成熟脐血来源树突状细胞 (CBDC )可以引发强大抗肿瘤免疫反应 ,CBDC培养 12d后用电镜分析形态 ,用流式细胞仪检测其表型。在不同培养时间 ,用3 H TdR掺入法检测CBDC和细胞因子诱导杀伤细胞 (CIK )的扩增功能 ;用MTT法检测被CBDC激活的CIK抗肿瘤活性。结果表明 ,体外诱导出形态典型的DC ,高表达主要组织相容性抗原I、II类分子、CD5 4 ,也表达CD80、CD83、CD1a,不表达CD6 8。体外培养 12d成熟的CBDC ,能使CIK以最高的比率扩增 ,2种细胞最佳混合比为 1∶10时被激活的CIK杀伤BEL 74 0 2肝癌细胞的功能最强 ,最佳效靶比为 80∶1     

杀伤性树突状细胞的研究进展

目的：探讨杀伤性树突状细胞在人体免疫功能中的作用。方法：在Pubmed上查阅杀伤性树突状细胞的相关文献,对其在啮齿类动物和人类体内的免疫作用进行归纳总结。结果：啮齿类和人类体内均有杀伤性树突状细胞,它可以直接连接固有免疫和适应性免疫,并具有杀伤肿瘤细胞的功能。结论：杀伤性树突状细胞可以为临床肿瘤免疫疗法提供一条新思路。     

树突状细胞在狼疮性肾炎肾组织中的分布及其临床意义

经肾活检和临床资料确诊的LN患者 5 3例 ,采用免疫荧光双标记染色与低照度荧光图像分析检测方法 ,观察到LN患者肾组织中树突状细胞分布的面积、数量及密度均明显增多。树突状细胞主要分布于肾小管间质和肾血管 ,而肾小球基本未见分布。此外 ,树突状细胞在肾小管间质分布程度与肾小管间质病变程度及肾功能呈正相关。提示树突状细胞可能参与了狼疮性肾炎的发病机制 ,并与患者肾小管间质病变及肾功能密切相关     

Vinpocetine Inhibited the CpG Oligodeoxynucleotide-induced Immune Response in Plasmacytoid Dendritic Cells

Plasmacytoid dendritic cells (pDCs) exert dual roles in immune responses through inducing inflammation and maintaining immune tolerance. A switch of pDC phenotype from pro-inflammation to tolerance has therapeutic promise in the treatment of autoimmune diseases. Vinpocetine, a vasoactive vinca alkaloid extracted from the periwinkle plant, has recently emerged as an immunomodulatory agent. In this study, we evaluated the effect of vinpocetine on phenotype of pDCs isolated from C57BL/6 mice and explored its possible mechanism. Our data showed that vinpocetine significantly downregulated the expression of CD40, CD80, and CD86 on pDCs and increased the expression of translocator protein (TSPO), the specific receptor of vinpocetine, in pDCs. Vinpocetine significantly inhibited the Toll-like receptor 9 signaling pathway and reduced the secretion of related cytokines in pDCs through TSPO. Furthermore, viability of pDCs was significantly promoted by vinpocetine. These findings imply that vinpocetine serves as an immunomodulatory agent for pDCs and may be applied for the treatment of pDCs-related autoimmune diseases.     

Development of Dendritic Cell-Based Immunotherapy for Autoimmunity

Dendritic cells are professional antigen-presenting cells that maintain immune tolerance to self-antigens by deleting or controlling the pathogenicity of auto-reactive T-cells. Dendritic cell-based immunotherapies show great promise for the restoration of tolerance in autoimmune disease. Dendritic cells can be modified ex vivo to induce stable tolerogenic function and be used as cellular ‘vaccines’ or they can be targeted in vivo with sophisticated antigen delivery systems. Tolerogenic dendritic cells induce antigen-specific T-cell tolerance in vivo and have therapeutic effects in animal models of autoimmunity. The current challenge is to bring tolerogenic dendritic cell therapy to the clinic.     

The effects of RelB deficiency on lymphocyte development and function

Multiple receptors that control cell growth and inflammation activate the NFκB pathway that comprises of two pathways. Dysfunction of the classical pathway leads to impaired adaptive and innate immunity in humans. In contrast the exact role of the alternative NFκB pathway mediated by RelB in humans remains largely elusive. We have recently identified deleterious mutations in RelB in patients with combined immunodeficiency and autoimmunity.We studied here the biological effects of RelB deficiency on the immune system. We show that the thymus in this patient is dysplastic and consequently new thymus emigrants are rare and there is an accumulation of CD45 RO⁺ T cells with an increase in CD62L⁺ central memory cells. The TCR repertoire of these cells appears skewed with selective clonal expansion. In vitro responses to T cell mitogens were markedly depressed and so were PHA induced IL2 and IFNγ production. In addition, the TH1 promoting T bet and STAT1 were reduced. In contrast, hyper-activation was seen in response to anti-CD3 and CD28. T cell dependent antibody responses were low to absent in all patients. We found that BAFF-R was reduced and CD40 signaling aberrant. Critically, CD27⁺ memory cells were absent.We have shown here for the first time the role of RelB on lymphocyte development in humans. In the absence of RelB, B cells development is arrested, resulting in poor production of immunoglobulins and specific antibodies. T cell maturation in the thymus appears altered with reduced output and production of a skewed T cell repertoire with expansion of clones which are likely the cause of the autoimmune features observed in these patients.     

The Role of Plasmacytoid and Myeloid Dendritic Cells in Induction of Asthma in a Mouse Model and the Effect of a TLR9 Agonist on Dendritic Cells

Purpose

To determine the role of plasmacytoid dendritic cells (pDC) and myeloid dendritic cells (mDC) in priming effector T cells to induce allergy, and to evaluate the effect of immunostimulatory sequences (ISS, TLR9 agonist) on dendritic cells.

Methods

Cultured mDC and pDC with/without ISS were injected intratracheally into sensitized Balb/C mice. Mice were sacrificed, and then pulmonary function tests, bronchoalveolar lavage (BAL), cell counts, and cytokine levels were evaluated. Migration of dendritic cells was also evaluated after ISS administration.

Results

In mice injected with mDC, airway hyperresponsiveness, eosinophil counts, and Th2 cytokine levels in BAL increased with increasing numbers of mDC injected. However, in mice injected with pDC, none of these changed, suggesting poor priming of T cells by pDC. In addition, mDC pulsed with ISS inhibited asthmatic reactions, and ISS administration inhibited migration of DC to the lung.

Conclusions

We suggest that pDC played a limited role in priming T cells in this asthma model and that mDC played a major role in inducing asthma. In addition, ISS inhibited migration of DC to the lung.     

IL-4对DC产生IL-12影响的研究

为了研究白细胞介素 4 (IL 4 )对树突状细胞 (dendriticcell,DC )产生白细胞介素 1 2 (IL 1 2 )p70的调节作用及其机制 ,将小鼠骨髓细胞在含GM CSF和IL 4的培养液中培养 6d ,加入成熟诱导剂脂多糖 (LPS )或TNF α、CpG ODN继续培养 2d以获得成熟DC。流式细胞仪检测DC表面CD80和MHCII类分子 ,混合淋巴细胞反应检测DC促进同种异体T细胞增殖的能力 ,ELISA法检测不同诱导剂诱导DC产生IL 1 2p70的能力 ,RT PCR和荧光定量PCR检测IL 1 2p35和p4 0mRNA的表达及其变化。结果显示小鼠骨髓细胞在含GM CSF、IL 4和成熟诱导剂的培养液中培养后可以获得成熟的DC ,成熟DC高表达CD80和MHCII类分子 ,具有较强的刺激同种异体T细胞增殖的能力 ,LPS、CpG ODN、TNF α、PolyI:C在促进DC成熟的同时能诱导DC产生有活性的IL 1 2 ,IL 4对IL 1 2的产生具有明显的促进作用 ,RT PCR和荧光定量PCR结果显示LPS诱导IL 1 2产生以及IL 4对其的促进作用与IL 1 2p35基因的转录水平增高有关