负载乳腺癌抗原的脐血树突细胞外泌体抗肿瘤免疫作用

目的:探讨负载乳腺癌抗原的人脐带血树突状细胞(DCs)分泌的外泌体的抗肿瘤免疫效应。方法:提取新鲜脐血单个核细胞,体外诱导分化为DCs,提取乳腺癌细胞MCF-7肿瘤抗原,并冲击脐血DCs,加入LPS诱导其成熟,利用流式细胞仪检测表型;超速离心法收获负载抗原的脐血DC外泌体;MTT法检测外泌体诱导的T淋巴细胞增殖反应及其激活的CTL对肿瘤细胞的杀伤效应。结果:脐血DC表面标志CD34、CD80、CD86、CD11c阳性细胞数与新鲜脐血单个核细胞相比明显增加(P<0.05);Western-Blot结果显示,负载乳腺癌的脐血DC外泌体携带有MHC-Ⅱ类、CD40、CD80、CD86分子;成熟脐血DC组及负载乳腺癌抗原脐血DC外泌体对同种异体来源的T细胞均有明显促增殖作用;负载乳腺癌抗原的脐血DC及其外泌体激活的CTL均能对乳腺癌细胞MCF-7产生显著地杀伤作用(P<0.05)。且在效靶比100∶1时,外泌体的杀伤作用高于负载抗原的脐血DC(P<0.05)。结论:负载乳腺癌抗原脐血DC分泌大量外泌体,可促进同种异体T细胞增殖,并能诱导特异性抗乳腺癌的免疫效应。     

人外周血未成熟树突状细胞的体外扩增和鉴定及抗成熟特性的研究

目的建立体外诱导、扩增具有抗成熟特性的未成熟树突状细胞(DC)的方法。方法从健康成人外周血分离单个核细胞(PBMC)，以rhGM—CSF和rhIL—4体外培养9d，于第7d加入rhIL—10，收集悬浮细胞，电镜观察形态，流式细胞仪测定细胞表型，并进行混合淋巴细胞反应(MLR)，观察其刺激同种异体未致敏T淋巴细胞增殖的能力。将培养的细胞分别用LYS、TNF—α继续刺激2d，再进行MLR，观察结果。结果PBMC经rhGM—CSF rhIL-4诱导并经rhIL-10处理后获得的细胞具有典型的DC形态特征，表达DC的特异性标志CD1a，不表达DC的成熟标志CD83，与对照组相比其共刺激分子CD86、CD40的表达显著下降，MLR示不能刺激同种异体T细胞增殖。经LPS、TNF—α刺激后亦不能刺激T细胞增殖。结论与rhIL—10联合培养可获得具有抗成熟特性的未成熟DC，这对于诱导大面积深度烧伤病人的异体皮移植免疫耐受具有良好的应用前景。     

2，4-二硝基氯代苯对小鼠骨髓源树突状细胞膜表面分子的影响

[目的]筛选小鼠骨髓来源树突状细胞（DC）在化学物致敏性评价中的合适指标。[方法]将皮肤致敏物2，4-二硝基氯代苯（DNCB）和皮肤刺激物十二烷基磺酸纳（SDS）分别作用于小鼠骨髓来源DC，设立对照组，在不同的时段，用流式细胞仪检测DC膜表面分子CD80、CD86和MHCⅡ类分子。[结果]CD80在SDS染毒后，48h和60h时表达增幅最大，约335％，而DNCB组和对照组则平稳增加，最大增幅分别为50％和60％；CD86表达在DNCB染毒48h时达到最大值，增加约262％，而SDS组和对照组最大增幅分别为72％和66％，12h后趋于降低；MHCⅡ类分子表达则在3组中均出现大幅波动。[结论]CD86分子表达在化学物致敏性评价中有一定的应用前景。     

DC细胞在辐射损伤抗原递呈及T细胞活化中的作用

目的 利用体外细胞共培养技术模拟体内肺组织微环境,探索树突状细胞（DC）在辐射损伤细胞的抗原提呈作用。方法 ⁶⁰Co γ射线照射的小鼠肺上皮细胞（MLE-12）与骨髓来源DC和/或脾T淋巴细胞培养48 h,流式细胞术检测DC细胞共刺激分子CD80/86和抗原肽识别复合物MHC Ⅰ/Ⅱ表达水平,T细胞活化标志CD69/28/152表达水平以及CD4⁺和CD8⁺亚群细胞数。结果 ⁶⁰Co γ射线照射的MLE-12细胞凋亡率呈剂量依赖性增高,明显刺激DC细胞CD80/86和MHC II表达,但对T细胞无直接活化作用;6 Gy照射的MLE-12细胞与DC细胞和T淋巴细胞共培养48 h,T细胞CD69和CD28表达增加,CD4⁺和CD8⁺亚群细胞数均明显高于对照组,同时DC细胞出现CD86和MHCI特异性高表达。结论 辐射损伤细胞可刺激DC细胞抗原提呈功能,并对T细胞进行活化。     

血管紧张素转化酶抑制剂对大鼠树突状细胞功能的影响

目的研究动脉粥样硬化(AS)大鼠树突状细胞(DC)的功能状态及血管紧张素转化酶抑制剂(ACEI)的影响。方法大鼠30只分组饲养后,分离外周血单个核细胞(PBMC),在含粒细胞巨噬细胞集落刺激因子(GM-CSF)、白介素-4(IL-4)培养条件下制备DC。用流式细胞仪检测DC表面共刺激分子CD86(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。酶联免疫吸附法(ELISA)测定MLR上清液中细胞因子水平。结果与正常对照组比较,AS大鼠DC表面CD86的表达明显增高,对T淋巴细胞刺激的能力增强,致炎细胞因子(IL-1β、TNF-а)分泌增多。与AS组相比较,AS应用依那普利组的DC表面CD86的表达明显降低,对T淋巴细胞刺激的能力下降,致炎细胞因子(IL-1β、TNF-а)分泌减少。结论AS大鼠DC处于激活状态,DC可能参与了AS的发病。ACEI对大鼠DC功能有明显的抑制作用,可能是其抗AS的作用机制之一。     

共刺激分子CD86在急性髓性白血病细胞的表达及其临床意义

目的探讨共刺激分子CD80、CD86在急性髓性白血病细胞中的表达及其临床意义。方法采用细胞培养白血病细胞（HL60,U937,NB4及I（562）及流式细胞仪检测共刺激分子CD80、CD86在急性髓性白血病患者骨髓细胞中的表达及其在急性髓性白血病细胞HL-60细胞、U937细胞、NB4细胞和K562细胞的表达。结果CD80在急性髓性白血病骨髓细胞中的表达很低或不表达,CD86在急性髓性白血病骨髓细胞表达[（27．86±19．65）％]高于对照组[（1．21±0．13）％,t=3．55,P〈0．01]。其中CD86在M4型（48．65±21．92）％、M5型（39．25±18．67）％表达较高,但与对照组单核细胞表达（50．204-20．31）％比较差异均无统计学意义（P〉0．05）。CD86在HL-60细胞、U937细胞和NB4细胞的培养24h后表达分别为（30．624-5．35）％、（24．12±5．23）％、（21．25±3．78）％,与培养48h后表达[（29．43±4．67）％、（26．564-6．54）％、（23．21±6．98）％]比较差异均无统计学意义（P〉0．05）。CD80在HL一60细胞、U937细胞和NB4细胞的表达很低或不表达,K562细胞不表达共刺激分子CD80和CD86。结论CD86在HL-60细胞、U937细胞和NB4细胞和急性髓性白血病患者骨髓细胞中表达。     

碘对小鼠骨髓树突状细胞前体表型及功能影响

目的 探讨碘对小鼠树突状细胞(dendritic cell,DC)前体表型及功能的影响.方法 选雌性C57BL/6J小鼠40只,按碘摄入量随机分为适碘组(NI)、低碘组(LI)、10倍碘过量组(10HI)、50倍碘过量组(50HI)4组,喂养8个月后检测小鼠DC表面分子表达和培养上清中白细胞介素-12(interleukin-12,IL-12)水平以及DC刺激T细胞的增殖能力.结果 LI组小鼠DC表面分子CD40+CD11c+、CD80+CD11c+、CD86+CD11c+、MHC-Ⅱ+CD11c+在CD11c+细胞中所占比例均低于NI组小鼠,差异均有统计学意义(P<0.05);50HI组小鼠DC表面分子CD40+CD11c+、CD80+CD11c+、CD86+CD11c+、MHC-Ⅱ+CD11c+在CD11c+细胞中所占比例均高于NI组小鼠,差异均有统计学意义(P<0.05);LI组小鼠DC培养上清中细胞因子IL-12浓度为(275.8±32.6)pg/mL,低于NI组小鼠的(548.7±51.3)pg/mL(P<0.01);50HI组小鼠DC培养上清中细胞因子IL-12浓度为(963.2±83.4)pg/mL,高于NI组小鼠(P<0.01);LI组的刺激指数(SI)为(5.23±1.96),低于NI组小鼠的(8.61±2.15)(P<0.05);50HI组小鼠的SI为(12.38±2.61),高于NI组小鼠(P<0.05).结论 摄入一定水平的碘可诱导小鼠DC前体的增殖和成熟,提高机体的免疫功能.     

HSP90多肽复合物对树突状细胞CD86、CD11c及IL-12p70表达的影响

目的研究HSP90多肽复合物对树突状细胞（dendritic cells,DC）功能的影响,并探讨HSP90多肽复合物修饰的DC疫苗对肿瘤的抑制作用。方法 HSP90多肽复合物刺激DC前后,ELISA法检测IL-12P70的表达水平,流式细胞仪检测CD86及CD11c的表达。建立C57BL/6小鼠Lewis肺癌模型,观察不同组别DC疫苗对肿瘤生长的抑制作用。结果 DC经HSP90多肽复合物刺激后,CD86及CD11c的表达上调（94.49±1.49及81.87±1.64）%,IL-12p70分泌量（97.11±5.26）pg/ml,均高于肿瘤粗提取物刺激DC组及未刺激空白DC组（P〈0.05）。HSP90多肽复合物修饰的DC疫苗可明显抑制肿瘤生长,抑制率为57.56%,凋亡率为26.35%,均明显高于对照组（P〈0.05）。结论 HSP90多肽复合物能够诱导DC成熟,经其修饰的DC能抑制肿瘤生长,可能与其能诱导肿瘤细胞凋亡有关。     

特发性血小板减少性紫癜患者外周血淋巴细胞共刺激分子的表达及白介素18的作用

目的本研究探讨特发性血小板减少性紫癜（ITP）患者外周血淋巴细胞共刺激分子CD80、CD86及其配体CD28的表达水平及白介素18的作用。应用免疫荧光标记和流式细胞技术检测34例ITP患者和34名正常人外周血淋巴细胞共刺激分子CD80、CD86及其配体CD28的表达，酶联免疫（ELISA）法测定血浆IL18。结果ITP患者外周血淋巴细胞CD80、CD86表达率（4．21&#177;2．27％，7．19&#177;5．16％）均明显高于正常对照组（2．34&#177;0．87％，4．08&#177;1．96％，P〈0．01）。ITP组血浆IL18含量为（538．31&#177;111．33）pg／ml，正常对照组血浆IL18含量为（489．44&#177;49．07）pg／ml。IL18含量与血小板数量呈显著性负相关（r=-0．395，P〈0．05）。结论ITP患者CD80和CD86共刺激分子过度表达，患者血浆中IL18水平比正常对照组明显升高（P〈0．05），其刺激分子CD80、CD86与ITP发病密切相关，IL18在ITP发病机理中起重要作用.     

氧化低密度脂蛋白对树突状细胞表面CD36表达的影响

目的探讨氧化低密度脂蛋白（OX—LDL）对树突状细胞表面CD36表达的影响。方法梯度离心法分离人外周血单核细胞,经含重组人粒细胞-巨噬细胞集落刺激因子和重组人白介素4的DC完全培养液中培养,使其分化为DC。DC分别与不同浓度OX—LDL（分别为10μg／ml、50μg／m1）共同孵育48h后,镜下观察DC形态变化,同时采用流式细胞术检测DCs表型（CDlot、CD83）和CD36的表达,以及用ELISA法检测细胞土清液中IL-10、IL—12含量。结果与对照组相比,OX—LDL50μg／ml组DCs表型CD1α、CD83以及CD36的表达明显上调（P〈0．01）,OX—LDL50μg／ml组细胞上清液中IL-10、IL—12含量明显分别明显升高和降低（P〈0．01）。结论DCs表面CD36的表达与OX—LDL诱导的DCs成熟以及炎症因子的释放密切相关,CD36可能在DCs分化成熟以及抗原递呈过程中发挥重要作用。     

慢性乙型肝炎患者外周血中树突状细胞的分离、纯化与扩增

目的探讨从慢性乙型肝炎(CHB)患者外周血中分离、纯化与扩增树突状细胞(DC)的有效方法。方法分别从健康自愿者(10例,对照组)和CHB患者(10例,实验组)外周血中分离出单核细胞(Mo),然后将实验组的Mo与GM-CSF、IL-4共同培养,于倒置显微镜下观察计数细胞,以流式细胞仪检测培养前、后的DC数量及其表面CD40、CD80、CD86和MHC-DR的表达水平,并与对照组比较。结果与对照组相比,实验组DC表面CD40、CD80、CD86和MHC-DR表达水平较低(P<0.01);经GM-CSF、IL-4联合培养5d后,其Mo中的DC数较培养前明显增多(P<0.01),且DC表面CD40、CD80、CD86和MHC-DR表达水平较培养前明显增高(P<0.01),与对照组相比则无明显差异(P>0.05)。结论GM-CSF、IL-4联合应用能有效地从CHB患者外周血中制备出大量具有功能活性的DC。     

Characterization of poly(D,L-lactic-co-glycolic acid) based nanoparticulate system for enhanced delivery of antigens to dendritic cells

Biodegradable nanoparticles made of poly(D,L-lactic acid-co-glycolic acid) (PLGA) copolymer were characterized for enhanced delivery of antigens to murine bone marrow derived dendritic cells (DCs) in vitro. PLGA nanoparticles were efficiently phagocytosed by the DCs (CD11c+, MHC class II+, CD86+) in culture, resulting in their intracellular localization. The efficiency of the uptake was influenced by the incubation time and nanoparticle concentration. DCs pulsed with PLGA nanoparticles containing an immunomodulator, monophosphoryl lipid A (MPLA), showed upregulation of surface expression of MHC class II and CD86 molecules. Delivery of a cancer-associated antigen (MUC1 mucin peptide: BLP25) and MPLA in PLGA nanoparticles was shown to be superior to their delivery in the soluble form for activation of na?ve T cells of normal and MUC1-transgenic mice. These results strongly suggest that PLGA nanoparticles provide an efficient vaccine delivery system for targeting DCs and the development of DC based cellular vaccines.     

Induction of cytotoxic T lymphocyte responses by cholera toxin-treated bone marrow-derived dendritic cells

Cholera toxin (CT), a powerful mucosal adjuvant, is a potent inducer of Th2-type responses via activation of co-stimulatory molecules for the induction of IgA antibody responses. Less appreciated is the ability of CT to induce and regulate cytotoxic T lymphocyte (CTL) responses. In order to help for clarifying mechanisms underlying the CTL-inducing ability of CT, we have examined the effects of CT on dendritic cells (DCs) that could lead to the induction of cytotoxic CD8(+) T cells. When bone marrow-derived DCs (BM-DCs) were cultured with CT in vitro, B7-1 but not B7-2 molecules were significantly enhanced and allogenic CTL responses were induced. Also, increased numbers of IFN-gamma-secreting CD8(+) T cells were elicited when CT-treated BM-DCs were co-cultured with allogenic CD8(+) CTLs. Antibody blockade of B7-1 on CT-treated BM-DCs suppressed allogenic CTL responses, further indicating the importance of CT-induced B7-1 molecules on DCs for the acquisition of cytolytic function by CTL precursors. CD40 signaling was proven not necessary for the CT-induced CTL response since CT-treated CD40(-/-) BM-DCs developed CTL responses equivalent to those detected in CT-treated BM-DCs derived from normal mice. Our results suggest that CT-treated DCs are effective inducers of CD8(+) CTL, and this induction is mediated through CT's ability to enhance B7-1 expression on DCs.     

Pleurotus ferulae water extract enhances the maturation and function of murine bone marrow-derived dendritic cells through TLR4 signaling pathway

Dendritic cells (DCs) play important roles in the regulation of immune system, which link innate and adaptive immune responses. Mature DCs produced interleukin (IL)-12 promote optimal type 1 T helper (Th1) cells and cytotoxic T lymphocytes. The extracts of traditional herbal medicines have been shown to enhance immune responses through promoting the maturation and cytokine production of DCs. Here, we investigated the effects of Pleurotus ferulae water extract (PFWE) on the maturation and function of bone marrow–derived DCs (BM–DCs). Upon PFWE treatment, BM–DCs dose-dependently upregulated the expression of CD40, CD80, CD86 and MHC II and increased the production of IL-12, IL-6 and tumor necrosis factor (TNF)-α but not for IL-10, which is mediated by TLR4 signaling pathway, at least partially. The production of prostaglandin E2 (PGE2) in BM–DCs was decreased by the treatment of PFWE. Moreover, PFWE treatment decreased the expression of active caspase-3 but increased the expression of CCR7. PFWE treated DCs enhanced the proliferation of allogenic CD8⁺ T cells and the capacity of antigen presenting to autologous CD8⁺ T cells. The combination of PFWE and CpG–ODN further enhanced the maturation and function of murine BM–DCs. The results showed that PFWE could enhance the maturation and function of DCs through TLR4 signaling pathway and has additive effect when combined with CpG–ODN, suggesting that PFWE alone or combined with CpG–ODN could be used to enhance the immune responses.     

Theileria annulata transformation altered cell surface molecules expression and endocytic function of monocyte-derived dendritic cells

Theileria annulata is a protozoan parasite transmitted by ticks to cattle. The most important processes of T. annulata are the infection and transformation of host monocytes, which promote cell division and generate a neoplastic phenotype. Dendritic cells play an important role in the development of adaptive immune responses against parasites and are traditionally classified into four types. One type of dendritic cell derived from afferent lymph was successfully transformed by T. annulata in vitro in a previous report. However, whether the monocyte-derived dendritic cells could be transformed and how the endocytic function is affected by T. annulata infection were not yet known. Bovine dendritic cells (DCs) derived from blood CD14⁺ monocytes were cocultured with T. annulata sporozoites in vitro. On day 15 post infection, rounded and continuously proliferating cells were observed. The effect of this transformation on cell phenotype was studied using immunostaining and flow cytometry. After transformation, the cells maintained the expression of the DC-specific marker CD11c, but it was downregulated as were the expression of CD11b, CD14 and CD86. In contrast, CD205, CD45 and MHC class Ⅱ molecules were upregulated in transformed cells. The levels of CD172a, CD21, CD40 and CD80 expression were very low in the transformed cells (<1 %). However, the transformed cells maintained high expression levels of MHC Ⅰ (>99 %). In addition, the normal and transformed DCs were cocultured with OVA-FITC antigen to compare the differences of the endocytic functions between these two types of cells. The results revealed that the endocytic functions of MoDCs were significantly inhibited after transformation by T. annulata.     

慢性乙型肝炎患者树突状细胞分离培养及其功能测定

目的探讨树突状细胞（DCs）在慢性乙型肝炎发病过程中的功能特点，为进一步研究慢性乙型肝炎的发病机制提供科学依据。方法对10例慢性乙型肝炎患者、10例乙型肝炎病毒（HBV）携带者及10例正常对照者的外周血DCs进行体外培养，应用流式细胞技术检测DCs表面分子HLA-DR、CD80、CD86的表达，并采用酶联免疫吸附试验（ELISA）检测DCs上清液白细胞介素（IL）-12的水平，比较DCs在慢性乙型肝炎不同感染阶段的功能特点。结果培养7天的DCs在透射电镜下观察，DCs充满每个视野，细胞体积较大，细胞表面突起较丰富，胞浆内粗面内质网及溶酶体丰富，线粒体结构较清楚，核大而圆，核膜清晰，染色质分布较均匀。正常对照组的HLA-DR、CD80和CD86的表达率均〉54％，而慢性乙型肝炎及HBV携带者组上述DCs表面分子的表达率普遍降低，与正常对照组比较，差异有显著性（P〈0．01），慢性乙型肝炎与HBV携带者组之间的差异无统计学意义（P〉0．05）。培养第7天DCs上清IL-12的表达水平在慢性乙型肝炎、HBV携带者组明显低于正常对照组（P〈0．05），而慢性乙型肝炎与HBV携带者组之间无明显差异（P〉0．05）。结论慢性乙型肝炎患者及HBV携带者均存在DCs表型和功能的缺失。