载脂蛋白A-I对树突状细胞表型及功能影响

目的:探讨载脂蛋白A-I(ApoA-I)对人外周血树突状细胞(PBDC)及体外培养的单个核诱导的树突状细胞(MD-DC)的影响及其机制。方法:采用免疫磁珠法,直接分离PB-DC或通过GM-CSF,IL-4诱导生成MDDC,DC经ApoA-I,LPS或TNF-α刺激后,用流式细胞技术(FCM)检测树突状细胞表面协同刺激分子表达的变化及细胞的吞噬能力;酶联免疫吸附法(ELISA)检测DC分泌的细胞因子;CFSE法检测经刺激后DC对T细胞增殖的影响。结果:分离高纯度PBDC,并成功诱导生成未成熟MDDC;经FCM检测,ApoA-I刺激后的PBDC和MDDC膜表面CD83分子表达上调,同时MDDC表面的CD40、CD86及MHC-Ⅱ分子的表达均增强;吞噬能力减弱;IL-12和TNF-α的分泌增加;并且可以诱导Th细胞的增殖。结论:在体外ApoA-I可以诱导PBDC和MDDC的成熟、活化,刺激其分泌细胞因子,发挥其免疫应答中抗原呈递,促进Th分化的作用;通过该作用ApoA-I可能参与了动脉粥样硬化的发生,发展中的免疫应答。     

黄芪多糖对树突状细胞表型及功能成熟的影响

目的 通过体外试验研究黄芪多糖（Astragalus mongholicus, ASP） 对树突状细胞（dendritic cells，DC）功能调节的机制，为进一步阐明黄芪多糖的免疫学活性提供依据。方法 应用流式细胞仪检测技术、扫描电镜技术、酶联免疫吸附试验检测DC表型和功能的各种指标。结果 本实验应用小鼠骨髓来源的DC，通过体外试验证明了黄芪多糖能够提高DC表面分子CD11c和MHCⅡ的表达，并且呈黄芪多糖浓度依赖性；空白组DC的吞噬功能很强，LPS组DC和黄芪多糖处理组DC吞噬功能都明显下降；黄芪多糖能够促进DC白细胞介素-12（IL-12）的表达；电镜观察DC的超微结构，可见黄芪多糖处理组DC突起增多，形态上更加成熟。结论 本实验结果证实了黄芪多糖能促进小鼠骨髓来源的DC表型及功能的成熟。     

蛋氨酸脑啡肽对树突状细胞表型及功能的影响

目的:通过体外细胞株研究蛋氨酸脑啡肽(Methionine-enkephalin,MENK)对树突状细胞(Dendritic cell,DC)功能调节的机制,为进一步阐明蛋氨酸脑啡肽的免疫活性提供依据。方法:应用扫描电镜技术、酸性磷酸酶活性检测、流式细胞仪检测技术、酶联免疫吸附试验检测DC表型和功能的各种指标。结果:本实验应用小鼠髓系DC2.4细胞株,电镜观察DC的超微结构,可见10-12mol/L蛋氨酸脑啡肽处理组DC突起增多,形态上更加成熟;DC表面MHCⅡ、CD86和CD40的表达增加;酸性磷酸酶活性降低;DC白细胞介素12(IL-12)的表达增加(P0.05)。结论:本实验结果证明了适宜浓度的蛋氨酸脑啡肽促进小鼠髓系DC2.4的表型及功能的成熟。     

早期脂多糖干预对大鼠树突状细胞表型和功能的影响

目的探讨早期脂多糖(LPS)干预对大鼠树突状细胞(DC)表型和功能的影响。方法用rrIL-4和rrGM-CSF体外诱导骨髓前体细胞,成熟组于第6天加入LPS1μg/mL,干预组分别于第0、3、6天连续加入LPS1μg/mL,对照组不加LPS,第7天收集细胞。采用免疫细胞化学方法、流式细胞术、同种淋巴细胞刺激试验和RT-PCR进行表型和功能鉴定。结果与对照组相比,MHC-Ⅱ、CD80、CD86、IL-12的表达和刺激同种T淋巴细胞增殖的能力在成熟组明显增高而干预组明显降低(P<0.01),Dextran-FITC摄取能力在成熟组明显降低而干预组明显增高(P<0.01)。结论早期LPS干预可抑制大鼠DC成熟,获得纯度更高的未成熟DC,为进一步研究免疫耐受奠定了基础。     

丹参酮ⅡA 对树突状细胞表型的影响及对功能的调控

目的:提取小鼠骨髓树突状细胞(DCs),体外给予丹参酮ⅡA 干预,观察药物刺激后DCs 功能的改变,从而探讨丹参酮ⅡA 在免疫系统中的作用机制。方法:提取小鼠的骨髓DCs,体外给予10 ng/ ml GM-CSF 及IL-4 的完全培养液培养,并在第5 天,磁珠分选得到纯度90%以上的树突状细胞,体外给予一定浓度的丹参酮ⅡA 及LPS 刺激,收集细胞及上清,运用流式细胞技术检测DCs 表型,ELISA 方法检测细胞上清TNF-β、IL-12 含量变化,同种混合淋巴细胞反应检测树突状细胞刺激淋巴细胞增殖及分化的能力。结果:在丹参酮ⅡA 浓度为500 ng/ ml 时,药物对DCs 抑制作用达到最大,因此选取该浓度为实验作用浓度;即在500 ng/ ml 作用下,实验组与对照组相比,DCs 表达MHCⅡ、CD86 及CD80 水平均显著降低(P<0.05);实验组DCs 分泌的TNF-β及IL-12 含量均显著降低(P<0.05);实验组DCs 刺激淋巴细胞增殖反应能力明显降低(P<0.05);实验组DCs 刺激T 淋巴细胞分泌IL-4 含量明显高于对照组,IFN-β含量明显低于对照组(P<0.05)。结论:丹参酮ⅡA 可以通过降低LPS 诱导的DCs 成熟状态,来参与免疫系统或自身免疫性疾病的发生发展。     

HBsAg脉冲的树突状细胞对CIK细胞功能的影响

目的:探讨HBsAg脉冲的树突状细胞(DC),对细胞因子诱导的杀伤细胞(CIK)的增殖和杀伤作用的影响。方法:选择慢性乙肝患者23例,用常规方法分离外周血单个核细胞(PBMC),经HBsAg脉冲后培养为特异性的DC,用3H-TdR掺入法检测该细胞对CIK细胞增殖的刺激作用,用乳酸脱氢酶释放法检测特异性CIK细胞对HepG2215细胞的特异性杀伤作用。结果:HBsAg脉冲的DC对CIK细胞的增殖具有刺激作用。由HBsAg脉冲的DC所诱导的特异性CIK细胞对HepG2215细胞的特异性杀伤作用增强。结论:HBsAg脉冲的DC可增强CIK细胞的杀伤活性。     

黄芪多糖对浆细胞样树突状细胞功能及成熟的影响

目的:通过体外实验研究黄芪多糖(Astragalus polysaccharide,APS)对浆细胞样树突状细胞(Plasmacytoid dendritic cell,pDC)功能及成熟的影响,为进一步阐明黄芪多糖的免疫学活性提供依据。方法:从健康志愿者外周血分离单个核细胞,流式细胞仪分选pDC,加入0、50、100、200mg/L的黄芪多糖共孵育,24小时后收集上清液,应用酶联免疫吸附试验检测pDC分泌的IFN-α、TNF-α、IL-6量,5天后收集细胞,应用流式细胞仪检测树突状细胞(Dendritic cell,DC)表型CD11c、CD80、CD86阳性率、光镜观察DC的形态、扫描电镜观察DC的超微结构。结果:黄芪多糖显著提高pDC分泌的IFN-α、TNF-α、IL-6量,并且呈黄芪多糖浓度依赖性(P0.05);黄芪多糖可促进pDC向DC的分化和成熟,并呈黄芪多糖浓度依赖性(P0.05)。结论:黄芪多糖能够增强pDC的功能,并促进其向DC的分化和成熟。     

人外周血单核细胞来源树突状细胞的成熟诱导

目的：探讨诱导树突状细胞成熟的最优方法。方法：以细胞因子GM-CSF和IL-4体外诱导人单核细胞来源的树突状细胞，分别采用CD40L、LPS、TNF-α、细胞因子鸡尾酒法（TNF-α、IL-6、IL-1β、PGE2）诱导成熟，24小时后收获DCs以流式细胞仪检测其成熟表型CD80、CD83、CD86、HLA-DR和FITC-Dextran的内吞能力，ELISA法检测其IL-12的分泌，MTT法检测其刺激淋巴细胞增殖活性。结果：CD40L、LPS、TNF-α、鸡尾酒法均可诱导DCs的成熟，其中以鸡尾酒法诱导成熟的效果最优，CD83的表达率为66.91%（P〈0.05）；成熟DCsFITC-Dextran的内吞能力明显下降；成熟DCsIL-12分泌量明显高于未成熟DCs，其中鸡尾酒法诱导成熟的DCs的IL-12分泌量最高，成熟的DCs有较强的刺激淋巴细胞增殖能力。结论：细胞因子鸡尾酒法是诱导DCs成熟的最佳方法。     

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.     

中亚苦蒿提取物对树突状细胞成熟及功能的影响

目的:探讨中亚苦蒿硅胶柱分离组分（AEM-SC）对小鼠树突状细胞（DC）成熟及免疫功能的影响。方法:制备中亚苦蒿大孔吸附树脂70%乙醇洗脱组分,经硅胶柱进一步分离得到洗脱组分AEM-SC,并对其多糖、总黄酮、三萜类含量进行测定。流式细胞术检测AEM-SC对DC表面分子表达水平、抗原吞噬能力及刺激同种异体T细胞增殖能力的...     

雷公藤内酯醇对浆细胞样树突状细胞功能及成熟的影响

目的 通过体外试验研究雷公藤内酯醇(triptolide)对浆细胞样树突状细胞(plasmacytoid dendritic cell,pDC)功能及成熟的影响,为进一步阐明雷公藤内酯醇的免疫学活性提供依据.方法 从健康志愿者外周血分离单个核细胞,流式细胞仪分选pDC,加入0、5、10、30 μg/L的雷公藤内酯醇共孵育,24 h后收集上清液,ELISA检测pDC分泌的IFN-α、IL-6、TNF-α量,5 d后收集细胞,流式细胞仪检测树突状细胞表型CD11c、CD80、CD86阳性率,光镜观察DC的形态,扫描电镜观察DC的超微结构.结果 雷公藤内酯醇显著降低pDC分泌的IFN-α、IL-6、TNF-α,并呈雷公藤内酯醇浓度依赖性(P＜0.05);雷公藤内酯醇可抑制pDC向DC的分化和成熟,并呈雷公藤内酯醇浓度依赖性(P＜0.05).结论 雷公藤内酯醇能够降低pDC的功能,并抑制其向DC的分化和成熟.

Abstract：

Objective To explore the mechanism of immunomodulatory activity of triptolide on healthy volunteers peripheral blood mononuclear cells (PBMC)-derived plasmacytoid dendritic cells (pDCs). Methods Healthy volunteers-derived pDCs were sorted by flow cytometry, then incubated with triptolide (0, 5, 10, 30 μg/L). After 24 hours, we detected the concentration of IFN-α, IL-6, TNF-α using ELISA. After 5 days, the cultrural cells were collected and analyzed by flow cytometry, light microscope and electron microscope scanning. Results Triptolide-treated pDCs secreted lower level of IFN-α,IL-6 ,TNF-α, triptolide could inhibit pDCs differentiation to DCs which displayed more immature morphology and immunophenotypes than untreated-pDCs. Conclusion Triptolide could decrease the immune function of pDCs, inhibit differentiation and maturation of pDCs.     

MBL对LPS诱导树突状细胞成熟的调节作用

目的 探讨甘露聚糖结合凝集素对细菌脂多糖诱导的人外周血单核细胞来源树突状细胞（MoDC）成熟的影响。方法 从健康成人外周血分离能黏附塑料的单核细胞，在rhGM-CSF和rhIL-4条件下培养5d，然后在有或无LPS和不同质量浓度（10～100mg／L）天然人MBL条件下继续培养2d。用倒置显微镜观察DC的形态，以FACS分析DC的表型，用MTT法测定DC刺激同种异体T细胞增殖的能力，以酵母多糖颗粒吞噬试验评估DC的抗原摄取能力，用ELISA检测DC培养上清液中TNF-α和IL-12p40＋p70的含量。结果 MBL以剂量依赖方式下调LPS诱导人MoDC表面分子CD83和CD86表达，增强其摄取酵母多糖颗粒的能力，降低其激发初始T细胞增殖的能力，并抑制其LPS诱导的TNF-α和IL-12 p40＋p70分泌，但MBL质量浓度低至10mg／L时对LPS诱导DC成熟作用几无影响。结论 高质量浓度MBL能抑制LPS诱导的DC成熟过程，提示其可能在LPS引发疾病包括败血症或感染性休克中具有调节作用，亦提供了分析DC分化成熟有关信号途径的新手段。     

Infection and maturation of monocyte-derived human dendritic cells by human respiratory syncytial virus, human metapneumovirus, and human parainfluenza virus type 3

Human respiratory syncytial virus (HRSV), human metapneumovirus (HMPV), and human parainfluenza virus type 3 (HPIV3) are common, important respiratory pathogens, but HRSV has a substantially greater impact with regard to acute disease, long-term effects on airway function, and frequency of re-infection. It has been reported to strongly interfere with the functioning of dendritic cells (DC). We compared HRSV to HMPV and HPIV3 with regard to their effects on human monocyte-derived immature DC (IDC). Side-by-side analysis distinguished between common effects versus those specific to individual viruses. The use of GFP-expressing viruses yielded clear identification of robustly infected cells and provided the means to distinguish between direct effects of robust viral gene expression versus bystander effects. All three viruses infected inefficiently based on GFP expression, with considerable donor-to donor-variability. The GFP-negative cells exhibited low, abortive levels of viral RNA synthesis. The three viruses induced low-to-moderate levels of DC maturation and cytokine/chemokine responses, increasing slightly in the order HRSV, HMPV, and HPIV3. Infection at the individual cell level was relatively benign, such that in general GFP-positive cells were neither more nor less able to mature compared to GFP-negative bystanders, and cells were responsive to a secondary treatment with lipopolysaccharide, indicating that the ability to mature was not impaired. However, there was a single exception, namely that HPIV3 down-regulated CD38 expression at the RNA level. Maturation by these viruses was anti-apoptotic. Inefficient infection of IDC and sub-optimal maturation might result in reduced immune responses, but these effects would be common to all three viruses rather than specific to HRSV.     

The presence of interleukin-27 during monocyte-derived dendritic cell differentiation promotes improved antigen processing and stimulation of T cells

Dendritic cells (DCs) are potent antigen-presenting cells necessary to establish effective adaptive immune responses. The cytokine environment that exists at the time of DC differentiation may be an important but often ignored determinant in the phenotypic and functional properties of DCs. Interleukin-27 (IL-27) is a unique cytokine that has both inflammatory and immune suppressive activities. Although it can both promote and oppose activity of different T-cell subsets, mostly anti-inflammatory activity has been described toward macrophages and DCs. However, the specific effect of IL-27 during DC differentiation and how that may change the nature of the antigen-presenting cell has not been investigated. In this report, we show that IL-27 treatment during monocyte-derived DC differentiation enhanced the ability to process antigens and stimulate T-cell activity. DCs differentiated in the presence of IL-27 showed enhanced acidification of latex bead-containing phagosomes that was consistent with elevated expression of vacuolar-ATPases. This resulted in inhibition of intracellular growth of Staphylococcus aureus. In addition, the levels of MHC class II surface expression were higher in DCs differentiated in the presence of IL-27. Production of IL-12 was also significantly increased during S. aureus infection of IL-27-differentiated DCs. The net effect of these activities was enhanced CD4⁺ T-cell proliferation and T helper type 1 cytokine production. These findings are important to a wide number of immunological contexts and should be considered in the development of future vaccines.     

Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells

Bacterial DNA and immunostimulatory (i.s.) synthetic CpG-oligodeoxynucleotides (ODN) act as adjuvants for Th1 responses and cytotoxic T cell responses to proteinaceous antigens. Dendritic cells (DC) can be referred to as “nature's adjuvant” since they display the unique capacity to sensitize naive T cells. Here, we demonstrate that bacterial DNA or i.s. CpG-ODN cause simultaneous maturation of immature DC and activation of mature DC to produce cytokines. These events are associated with the acquisition of professional antigen-presenting cell (APC) function. Unfractionated murine bone marrow-derived DC and FACS^®-fractionated MHC class II^low (termed immature DC) or MHC class II^high populations (termed mature DC) were stimulated with bacterial DNA or i.s. CpG-ODN. Similar to lipopolysaccharide, i.s. CpG-ODN caused up-regulation of MHC class II, CD40 and CD86, but not CD80 on immature and mature DC. In parallel both DC subsets were activated to produce large amounts of IL-12, IL-6 and TNF-α. CpG-ODN-activated DC displayed professional APC function in allogeneic mixed lymphocyte reaction and in staphylococcal enterotoxin B-driven naive T cell responses. We interpret these findings to mean that bacterial DNA and i.s. CpG-ODN cause maturation (first step) and activation (second step) of DC to bring about conversion of immature DC into professional APC.     

VIP调节LPS激活的树突状细胞免疫功能

目的 研究神经肽VIP能否调节LPS激活的树突状细胞(DC)的免疫功能.方法 联合应用rm GM-CSF和rmIL-4自C57BL/10.A小鼠骨髓细胞制备DC;以LPS和/或VIP刺激DC;收集DC及其上清夜行ELISA分析;自DC提取总RNA行RNAse分析.结果 VIP明显抑制LPS激活的DC分泌细胞因子IL-2、IL-12和TNF-α,但对IL-6的抑制作用则不明显;VIP明显抑制LPS激活的DC表达化学因子MIP-2和-10,但对化学因子Rantes、MIP-1α和MIP-β的抑制作用并不明显.结论 VIP对LPS激活的DC的免疫功能有负性调节作用.     

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。     

小反刍兽疫病毒对鼠源树突状细胞成熟分化的影响

目的研究和探讨小反刍兽疫病毒(PPRV)对小鼠骨髓来源的树突状细胞(BM-DCs)成熟和分化功能的影响。方法从小鼠骨髓中分离得到DCs,经过IL-4和GM-CSF诱导成熟;流式细胞术检测DCs表面共刺激分子表达及吞噬FITC-Dextran的能力;ELISA检测细胞因子IL-6、IL-12p40、IL-1β和IL-10的表达水平。结果利用LPS和PPRV处理DCs 24 h,发现与阳性对照LPS组相比,PPRV作用后显著抑制了CD40和MHC-Ⅱ的表达(P<0.05);滴度适度的PPRV可以显著增加CD40、CD80和CD86的表达,显著升高IL-6和IL-10的分泌(P<0.05),吞噬FITC-Dextran的能力并无显著差异,各组之间也无影响,且不同比例的PPRV对DC存活率无显著影响(P>0.05)。结论 PPRV在一定滴度范围内具有潜在的促进小鼠DC成熟的功能,为免疫学研究奠定基础。