鸡尾酒法体外培养人外周血来源的树突状细胞

目的 探讨从健康人外周血中体外诱导培养成熟树突状细胞(DC)的方法.方法 用密度梯度离心法分离人外周血单个核细胞(PBMC),再用贴壁法获取单核细胞后加入人粒细胞-巨噬细胞集落刺激因子(GM-CSF)和IL-4,培养6 d后分组:Ⅰ组为对照组,仅含上述细胞因子;Ⅱ组加入多聚次黄嘌呤胞嘧啶核苷酸(Poly I∶C);Ⅲ组加入钙离子载体(CI)A23187;Ⅳ组加入Poly I∶C和CI A23187.第8天收获细胞,显微镜下观察形态,流式细胞术检测DC表型,体外同种混合淋巴细胞反应检测DC刺激T细胞的增殖活性.结果 Ⅳ组细胞形态学观察可见典型DC特征,荧光激活细胞分离器(FACS)检测高表达CD83、CD80、CD86和CD40;混合淋巴细胞反应(MLR)表明Ⅳ组细胞具有较强的刺激T细胞增殖能力.结论 PBMC体外经过细胞因子序贯、联合诱导培养能够获得大量功能成熟的DC.     

吡咯烷二硫代氨基甲酸酯对树突状细胞成熟的影响

目的：观察NF-κB抑制剂吡咯烷二硫代氨基甲酸酯（PDTC）对树突状细胞（DC）成熟的影响。方法：利用PDTC作用于体外培养的大鼠不成熟DC，细菌脂多糖（LPS）刺激后，流式细胞仪检测DC共刺激分子CD80、CD86、CD40表达；ELISA分析DC分泌IL—12的变化，NF—κB DNA结合ELISA检测核内NF—κB活性。结果：PDTC抑制DC共刺激分子CD80、CD86、CD40表达；减少DC分泌IL-12；抑制核内NF—κB DNA结合活性。结论：PDTC抑制DC成熟。     

大黄素对体外诱导人血来源树突状细胞的影响

目的探讨大黄素对体外诱导培养的人血来源树突细胞(DC)的影响。方法分离健康人外周血单核细胞,经培养后获得未成熟DC(iDC),重组人粒-巨噬细胞集落刺激因子(rhGM-CSF,106IU.L-1)和重组人白细胞介素4(rhIL-4,8×105IU.L-1)诱导获得成熟DC(mDC)。实验分为iDC组、mDC组和大黄素组,mDC组于d 5加入脂多糖(LPS,1 mg.L-1)刺激,大黄素组采用mDC在d 5经LPS刺激后于d 7加入大黄素(100 mg.L-1)共培养2 d。用倒置显微镜和电镜观察DC细胞形态,用流式细胞仪检测DC表面共刺激分子的表达水平。结果经rhGM-CSF、rhIL-4诱导和LPS刺激,培养9 d后可获得表面有丰富分叉状胞浆突起的毛刺状mDC。大黄素组DC表面突起短而少,呈iDC形态。大黄素组CD80、CD83和CD86表达率分别为(13.4±6.6)%、(9.3±2.2)%和(84.2±6.3)%,低于mDC组[分别为(39.3±8.6)%、(30.7±5.6)%和(95.4±3.2)%,P<0.01];大黄素组CD14表达率为(8.4±2.8)%显著高于mDC组的(3.7±2.3)%(P<0.01)。大黄素组HLA-DR及CD11c表达与mDC组比较无显著差异(P>0.05)。结论大黄素能干扰DC表面突起的形成和表面共刺激分子的表达。     

辛伐他汀对急性心肌梗死患者树突状细胞功能的影响

目的 研究急性心肌梗死（AMI）患者树突状细胞（DC）的功能及辛伐他汀对DC的影响. 方法 将32例AMI分为常规治疗组（R组）和常规治疗加辛伐他汀治疗组（R+S组）各16例,分别于治疗前及治疗后2周取血分离外周血单个核细胞,在含粒细胞巨噬细胞集落刺激因子和白细胞介素（IL）-4的培养条件下制备DC. 用流式细胞仪检测DC表面共刺激分子CD 86（B7-2）的表达;混合淋巴细胞反应（MLR）检测DC对同种异体T淋巴细胞的刺激能力;ELISA法测定MLR上清液中的细胞因子;探讨CD86 表达与冠心病危险因素及超敏C反应蛋白（hs-CRP）的相关性. 结果 与正常对照组比较,AMI患者DC表面CD86 的表达明显增高;对T淋巴细胞刺激的能力增强;经DC刺激的淋巴细胞分泌致炎细胞因子（IL-1β、IL-6、肿瘤坏死因子ɑ）增多,抑炎细胞因子（IL-10）减少;用药前CD86 的表达与血低密度脂蛋白胆固醇（LDL-C）水平正相关;辛伐他汀抑制DC功能的同时显著降低血hs-CRP水平;且CD86 与hs-CRP水平正相关. 结论 AMI患者DC功能亢进,由此启动的T淋巴细胞的增殖和炎性细胞因子分泌可能是AMI患者动脉斑块不稳定的因素之一;LDL-C可能是斑块不稳定的刺激因素;辛伐他汀抑制斑块炎症的机制之一是抑制DC.     

狼疮肾炎患者肾组织核因子-κB和外周血树突状细胞的表达

目的探讨狼疮肾炎（LN）肾核因子（NF）-κB（NF-κB）的表达和外周血树突状细胞（DCs）表面标志的改变。方法对40例LN患者肾组织,用碱性磷酸酶-小鼠抗碱性磷酸酶免疫复合物法（APAAP）测定NF—κB表达。采用密度梯度离心法分离外周血DCs。结果LN患者肾组织中,NF-κB在肾小球及肾小管均呈高水平的表达;他克莫司处理后的DCs较未处理组CD80^＋、CD86^＋、HLA—DR^＋表达下降;他克莫司处理前后对DCs的形态无明显影响;他克莫司处理后的DCs能明显抑制T细胞增殖及T细胞向Th2细胞转化。结论他克莫司在体外可抑制LN患者外周血DCs的成熟,NF—κB在LN肾组织局部的表达与LN的发生和发展有关。     

干扰素-α对慢性乙肝患者外周树突状细胞的影响

目的通过干扰素-α刺激体外培养的慢性乙肝患者外周血树突状细胞,检测树突状细胞CD80、CD86的表达,检测培养液中IL-12的浓度,以探讨干扰素对慢性乙肝患者外周血树突状细胞的影响,进而探讨干扰素治疗慢性乙肝的作用机理。方法抽取慢性乙肝患者外周血用Romani方法培养,对照组加入IL-4和GM-csf,实验组加入IL-4、GM-csf和干扰素-α培养;第9天时收集细胞应用流式细胞仪检测CD80、CD86;第12天时收集上清,用ELISA法检测IL-12的浓度。结果应用干扰素的试验组树突状细胞CD80、CD86的表达有所增加,上清中IL-12的浓度也较高。结论干扰素促进慢性乙肝患者外周树突状细胞表型成熟和功能增强。     

不同细胞因子诱导树突状细胞体外抗肿瘤效应研究

目的研究不同细胞因子诱导的树突状细胞(DC)体外抗肿瘤效应。方法常规分离健康人外周血单核细胞,分别采用粒巨噬集落刺激因子(GM-CSF),白细胞介素(IL)-4,肿瘤坏死因子(TNF),GM-CSF、IL-4、α干扰素(IFN-α)细胞因子组合将其诱导为DC,并分别负载SMMC7721细胞冻融抗原;用流式细胞仪检测第9天时细胞表面HLA-DR、CD1α、CD80、CD83的表达;采用四甲基偶氮唑盐比色(MTT)法检测各组刺激自体淋巴细胞增殖能力,并检测各组DC诱导的细胞毒性T淋巴细胞(CTL)对SMMC7721细胞株的杀伤率。结果第9天显微镜下观察各组细胞,其细胞形态无明显差异,但流式细胞仪显示IFN-α组DC其膜表面标志CD80、CD83、CD1a表达上调,在刺激同种T细胞增殖能力方面,较TNF-α组DC效果明显。且IL-12分泌明显增加。在肝癌细胞株SMMC7221杀伤实验中,培养液中加入IFN-α后杀伤效应明显。结论与TNF-α相比,IFN-α诱导DC一定程度上能增强其特异性杀伤能力。     

TNF-α和HBsAg对慢性乙型肝炎患者树突状细胞功能的调节作用

目的　研究肿瘤坏死因子 α(TNF α)和HBsAg对体外培养的慢性乙型肝炎 (慢性乙肝 )患者树突状细胞 (DC)功能的调节作用。方法　分离慢性乙肝患者外周血单核细胞 ,以人粒细胞巨噬细胞集落刺激因子 (GM CSF) +白细胞介素 4(IL 4)培养诱导DC。部分DC培养时加入TNF α(处理 1组 )或TNF α +HBsAg(处理 2组 ) ,于培养第 7天时 ,检测各组DC的表面分子CD1a和CD83的表达水平 ,刺激同种异体淋巴细胞增殖和诱导自体T细胞的细胞毒作用的能力 ,并测定各组DC培养上清中IL 6、IL 1 2的含量。结果　处理 1组DC的表型分子CD1a和CD83表达水平明显高于对照组 ,处理 2组DC的CD1a和CD83表达水平均显著高于对照组 ,并且与正常人群组无显著性差异。处理 1组DC刺激同种异体混合淋巴细胞反应的能力显著强于对照组 ,但较处理 2组弱 (P <0 0 5 )。各组DC诱导自体细胞毒性T细胞 (CTL)杀伤HepG2 2 2 1 5细胞的能力 ,处理 2组 >处理1组 >对照组 (均为P <0 0 1 ) ;DC诱导自体CTL对HepG2和K5 6 2细胞毒作用的能力 ,2个处理组均显著强于对照组。处理 2组的IL 6分泌水平显著低于对照组和处理 1组 ;2个处理组DC的分泌IL 1 2能力均显著高于对照组 ,且处理 2组明显高于处理 1组。结论　在体外诱导DC时 ,加入TNF α能有效地促进慢性乙肝     

TNF—α和IFN-α对人外周血树突状细胞功能活性的影响比较

目的研究不同细胞因子诱导的人外周血树突状细胞（dendritic cells,DC）其功能活性的不同。方法常规分离健康人外周血单核细胞,分别采用GM-CSWIL-4／TNFα、GM—CSWIL-4／细胞因子组合将其诱导为DC,用ELISA法检测各组细胞培养至第9天时其上清液中IL．12含量;用流式细胞仪检测第9天时细胞表面HLA—DR、CD1α、CD80、CD83的表达;采用MTT法检测各组刺激自体淋巴细胞增殖能力。结果第9天显微镜下观察各组细胞,IFN—α组DC细胞形态均一,有明显突起,FACM显示IFN—α组DC其膜表面标志CD1α、CD80、CD83表达上调,在刺激同种T细胞增殖能力方面,较TNF-α组DC效果明显。且IL-12分泌明显增加。结论与TNFα相比,IFN仪能在一定程度上增强其诱导的DC功能。     

小鼠骨髓来源树突状细胞对T细胞的活化作用

目的 从小鼠骨髓中体外诱导、扩增树突状细胞(DCs),检测DCs对T细胞的活化作用.方法 从小鼠骨髓中分离单个核细胞(MNCs),在重组鼠粒细胞-巨噬细胞集落因子(rmGM-CSF)、白细胞介素4(IL-4)和肿瘤坏死因子α(TNF-α)的诱导下培养扩增DCs.光学显微镜观察其形态特征,流式细胞仪分析其表型特征,混合淋巴细胞反应(MLR)检测DCs刺激同种异体T细胞增殖的能力.结果 MNCs经过rmGM-CSF、IL-4和TNF-α诱导培养12 d后,具有典型的DCs形态,高表达MHCⅡ类分子、CD11c、CD80、和CD86抗原.具有极强的激发刺激同种异体T细胞增殖的能力.结论 体外诱导、扩增DCs具有极强的激发刺激同种异体T细胞增殖的能力.     

中介素通过激活AMPK信号通路对脂多糖诱导巨噬细胞极化的影响

目的探讨中介素(intermedin,IMD)对脂多糖(lipopolysaccharide,LPS)诱导小鼠单核巨噬细胞系RAW 264.7极化的影响及其作用机制。方法RAW 264.7细胞随机分为对照组、LPS组、LPS+IMD组、LPS+IMD+CC(AMPK抑制剂Compound C)组。Real time-PCR法检测TNF-α、CD86、iNOS、Arg^-1、CD206 mRNA表达,Western blot法检测p-AMPK、AMPK、TNF-α、IL-6和IL^-10蛋白表达,流式细胞术检测巨噬细胞亚型,ELISA法检测培养基上清IL-6和TNF-α浓度。结果与对照组及LPS组比较,IMD处理可增加AMPK磷酸化水平,增加p-AMPK/AMPK比值;与对照组相比,LPS诱导可导致巨噬细胞发生M1极化,M1型标志分子CD86、TNF-α及iNOS mRNA表达升高,M2型标志分子CD206、Arg^-1 mRNA表达降低,上调促炎因子TNF-α、IL-6表达,降低抑炎因子IL^-10表达,使M1型细胞数量增加,细胞上清中TNF-α、IL-6分泌增加;而IMD处理可抑制LPS诱导的M1极化,AMPK抑制剂Compound C组处理可在一定程度上拮抗这一作用。结论IMD通过激活AMPK信号通路抑制LPS诱导的巨噬细胞M1型极化。     

吡格列酮对脂多糖刺激的星形胶质细胞白介素-1β的抑制作用及机制

目的研究吡格列酮(Pio)对脂多糖(LPS)诱导的星形胶质细胞白介素-1β(IL-1β)抑制作用及JNK信号传导通路对其的影响。方法 ELISA法测定培养液中IL-1β的含量;免疫荧光染色法观察星形胶质细胞IL-1β的蛋白表达;Western blot检测星形胶质细胞磷酸化JNK1和磷酸化c-Jun蛋白表达水平的改变。结果星形胶质细胞在LPS(10 mg.L-1)刺激下IL-1β的含量、蛋白表达以及磷酸化JNK1、磷酸化c-Jun蛋白水平与正常对照组比较均增高。特异性JNK特异性阻断剂SP600125(10.0μmol.L-1)可明显抑制LPS引起的IL-1β产生增加;Pio(0.1、1.0、10.0μmol.L-1)则可降低IL-1β产生,抑制IL-1β的蛋白表达及下调p-JNK1、p-c-Jun蛋白水平。结论 Pio能明显抑制LPS诱导的大鼠皮层星形胶质细胞IL-1β表达增加,这种作用可能与其下调JNK信号转导通路有关。     

G1-4 A,an arabinogalactan polysaccharide from Tinospora cordifolia increases dendritic cell immunogenicity in a murine lymphoma model

The immunogenicity of dendritic cells (DC) is known to increase with their maturation state and both are induced by microbial products like LPS. In this study, we have investigated the effect of G1-4A, a polysaccharide isolated from Indian medicinal plant, Tinospora cordifolia on phenotypic and functional maturation of murine bone marrow derived dendritic cells (BMDC) and its ability to be used as an adjuvant in immunotherapy. G1-4A, enhanced surface expression of CD40, CD80, CD86, MHCII by BMDC in vitro and splenic DC in vivo. T cell allostimulatory activity and secretion of IL-12 and TNFα by BMDC were also increased. Treatment with G1-4A resulted in decreased phagocytosis and increased antigen processing that are characteristic of mature DC. G1-4A treated DC cross presented exogenous antigens on a MHC I background which resulted in the activation of cytotoxic T cells. These cells thus activated could cause lysis of target tumor cells in vitro. Administration of tumor lysate pulsed G1-4A treated DC resulted in decreased tumor burden in preventive as well as therapeutic tumor challenge experiments in a murine lymphoma model. These results thus confirm that G1-4A could be a promising nontoxic maturation agent to be potentially used in DC based immunotherapy of tumor.     

Effect of pentoxifylline on differentiation and maturation of human monocyte-derived dendritic cells in vitro

Pentoxifylline (PTX) is a drug used for the treatment of vascular disorders, but it also has a positive therapeutic effect in experimental models of some autoimmune diseases. In this work, we studied the effect of PTX on human monocyte-derived dendritic cells (MDDCs). Immature MDDCs were generated in vitro from monocytes in the presence of recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF) and recombinant human interleukin-4 (rhIL-4), while mature MDDCs were obtained by cultivation of immature MDDCs with lipopolysaccharide (LPS). PTX (200 micro g/ml) was added at the beginning of cell cultivation. We found that PTX significantly impaired differentiation and function of immature MDDCs, as judged by the reduced allostimulatory activity of these cells on allogeneic T cells and down-regulation of costimulatory and adhesion molecules, such as CD86, CD40 and CD54. The maturation of MDDCs in the presence of PTX and LPS was characterized by the decreased expression of maturation marker CD83 and costimulatory molecule CD86, as well as lower stimulation of alloreactive T cells compared to the control MDDCs cultivated with LPS alone. PTX-treated MDDCs which were induced to mature with LPS produced lower levels of TNF-alpha, IL-12 and IL-18 and higher levels of IL-10 than corresponding control MDDCs. PTX did not significantly alter endocytosis of dextran by both immature and mature MDDCs. Cumulatively, our results show for the first time that PTX might impair differentiation, maturation and function of human MDDCs in vitro, suggesting an additional mechanism of its immunomodulatory activity.     

阿奇霉素对支气管哮喘患儿外周血树突状细胞功能的影响

目的:观察阿奇霉素对支气管哮喘患儿外周血单个核细胞(PBMC)来源树突状细胞(DCs)功能的影响,探讨阿奇霉素免疫调节作用的机制.方法:选择16例支气管哮喘发作期患儿为研究对象,18例健康儿童为对照组.无菌条件下采用密度梯度离心法获取PBMC,体外经rhGM-CSF、rhIL-4诱生DCs.哮喘组置于不同浓度阿奇霉素(...     

Effects of vasoactive intestinal peptide on phenotypic and functional maturation of dendritic cells

The present study was designed to investigate the effects of vasoactive intestinal peptide (VIP) on differentiation, maturation of dendritic cells (DCs) in vitro. DCs were derived from the murine bone marrow hemopoietic progenitor cells by culturing in RPMI 1640 complete medium supplemented with GM-CSF and IL-4 in the presence or absence of various concentrations of vasoactive intestinal peptide (VIP) and lipopolysaccharide (LPS). The phenotype of DCs was analyzed by flow cytometry. Mixed leukocyte reaction (MLR) was employed to measure the capacity of DC to stimulate the allogeneic T cells. IL-12p70 secretion by DC was examined by ELISA. In the absence of LPS, VIP, in a dose dependent manner, up-regulated the expression of CD80, CD86, CD54 and CD40, but down-regulated the expression of MHC class II molecule (Ia(b)). In the presence of LPS, VIP also dose dependently up-regulated the expression of CD80, CD86, CD54 and CD40, and down-regulated the expression of Ia(b). The capacity to stimulate alloreactive T cells and the production of IL-12p70 by DC were significantly augmented by VIP when compared with VIP-untreated DCs. These data suggest that VIP could promote the phenotypic and functional maturation of DCs, hereby regulating the type and outcome of the conducting immune response.     

紫草素通过TLR4/NF-κB信号通路抑制由脂多糖诱导的巨噬细胞炎症研究

目的 研究紫草素抑制由脂多糖(LPS)诱导的巨噬细胞炎症的机制.方法 以淀粉培养基注射BALB/c小鼠腹腔,诱导并分离巨噬细胞,以APC标记F4/80染色,并用流式细胞仪检测分离巨噬细胞情况;用1 mg·L-1的LPS刺激上述分离的巨噬细胞,分组如下:DMSO溶剂对照组,LPS对照组,LPS+低剂量紫草素组(0.1 μmol·L-1);LPS+中剂量紫草素组(1 μmol·L-1);LPS+高剂量紫草素组(10 μmol·L-1);通过ELISA和实时定量PCR(qRT-PCR)方法分别测定各处理组培养上清液以及细胞中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)和白细胞介素-10(IL-10)的表达情况;Western blot分别测定Toll样受体4(TLR4)和核因子-κB(NF-κB)的p65亚基磷酸化表达情况.结果 流式细胞检测可知,APC标记的F4/80染色巨噬细胞的纯度可达97.8%;ELISA和实时定量PCR结果显示,紫草素处理后可以抑制由LPS刺激细胞因子TNF-α、IL-1β和IL-6的表达(P<0.05),并增加IL-10的表达(P<0.05),且呈现出一定的浓度依赖性;Western blot结果显示,紫草素能下调由LPS刺激的TLR4的表达水平和NF-κB的p65亚基磷酸化表达.结论 紫草素的抗炎机制可能是通过TLR4介导的信号通路活化NF-κB,抑制IL-1β、IL-6和TNF-α的分泌,并促进IL-10的分泌.     

Fatty acids isolated from royal jelly modulate dendritic cell-mediated immune response in vitro

Royal jelly (RJ), especially its protein components, has been shown to possess immunomodulatory activity. However, almost nothing is known about the influence of RJ fatty acids on the immune system. In this work we studied the effect of 10-hydroxy-2-decanoic acid (10-HDA) and 3,10-dihydroxy-decanoic acid (3,10-DDA), isolated from RJ, on the immune response using a model of rat dendritic cell (DC)-T-cell cocultures. Both fatty acids, at higher concentrations, inhibited the proliferation of allogeneic T cells. The effect of 10-HDA was stronger and was followed by a decrease in interleukin-2 (IL-2) production and down-regulation of IL-2 receptor expression. Spleen DC, cultivated with 10 microg/ml of fatty acids down-regulated the expression of CD86 and the production of IL-12, but up-regulated the production of IL-10. In contrast, DC, pretreated with 100 microg/ml of 3,10-DDA, up-regulated the expression of CD86 and augmented the proliferation of allogeneic T cells. The highest dose (200 microg/ml) of both fatty acids which was non-apoptotic for both T cells and DC, down-regulated the expression of MHC class II and CD86, decreased the production of IL-12 and made these DC less allostimulatory. The immunosuppressive activity of 3,10-DDA was also confirmed in vivo, using a model of Keyhole lymphet hemocyanine immunization of rats. In conclusion, our results showed the immunomodulatory activity of RJ fatty acids and suggest that DC are a significant target of their action.