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。     

树突状细胞对肿瘤细胞株的直接杀伤活性

目的 对比分析干扰素－γ（Interferon-γ,IFN-γ)或脂多糖（lipoplysaccharide,LPS)刺激后的树突状细胞（dendritic cell,DC)对肿瘤细胞杀伤活性的差异。方法 分离健康供者外周血单核细胞，用粒单细胞集落刺激因子和白介素－4诱导为DC。于培养液中加入LPS或IFN－γ培养12h，作为LPS激活的DC（LPS－DC）及IFN－γ激活的DC（IFN－DC）。用流式细胞仪检测DC表面共刺激分子的改变，以明确LPS或IFN－γ对DC的不同刺激作用；同时，以恶性血液病细胞株HL－60 Jurkat及Daudi为靶细胞，用不同效靶比与DC共同培养18h，采用^51Cr释放试验检测LPS－DC及IFN－DC抗肿瘤活性的差异。结果 ①LPS及IFN－γ可不同程度的上调DC表面CD86、CD80、CD83及CD1a的表达，以LPS刺激组明显。②IFN－γ和LPS可分别增强DC对HL60及Daudi的杀伤活性，在效靶比为20：1及10：1时杀伤率与未加刺激因子对照组（medium-DC)相比差异有显著意义（P＜0．05）。相反，IFN－γ－DC对Daudi、LPS－DC对HL－60无明显杀伤活性，但两者对Jurkat均具杀伤作用。结论 LPS及IFN－γ激活的DC对肿瘤细胞的杀伤活性具有相对肿瘤特异性。     

地尔硫抑制酶修饰低密度脂蛋白对外周血中树突状细胞分化成熟的影响研究

目的:探讨酶修饰低密度脂蛋白(E-LDL)对外周血中树突状细胞(DC)分化成熟的影响以及地尔硫对其的影响作用。方法:梯度离心法分离人外周血单核细胞,用含重组人粒细胞-巨噬细胞集落刺激因子和重组人白介素4的Cellgro培养液培养5d,使其分化为DC。分别用E-LDL、地尔硫加E-LDL刺激DC48h后,镜下观察DC的形态,采用流式细胞术检测DC表型(CD1a、CD80、CD86、HLA-DR)的表达,并设磷酸缓冲盐对照组进行比较。结果:光镜下DC形态无明显变化,各组DC的CD1a、CD80、CD86和HLA-DR的表达有显著性差异(P<0.05),E-LDL处理后DC表面抗原的表达较对照组升高,地尔硫加E-LDL处理后DC表面抗原的表达较E-LDL组下调。结论:E-LDL可促进DC的分化成熟,地尔硫对E-LDL激活DC具有抑制作用。     

曲古菌素A诱导淋巴瘤Raji细胞表达共刺激分子CD80及CD86的表达

目的探讨组蛋白去乙酰化酶抑制剂曲古菌素A(TSA)诱导淋巴瘤细胞Raji细胞表达共刺激分子CD80和CD86及其与免疫应答的关系。方法采用MTT法和倒置相差显微镜观察不同浓度TSA对Raji细胞的抑制作用,流式细胞仪检测150 nmol/L TSA作用Raji细胞后的细胞表达共刺激分子CD80和CD86及细胞活力,RT-PCR分析CD80 mRNA和CD86 mRNA表达情况。结果 TSA对Raji细胞的抑制作用具有时间和剂量依赖性,150 nmol/L TSA作用后可上调Raji细胞表达CD80,并对Raji细胞有直接杀灭作用。结论 TSA可以诱导Raji细胞表达共刺激分子CD80,不表达CD86,促进细胞的免疫应答,为抗恶性血液病提供了一个新的免疫治疗方法。     

他克莫司对体外培养的大鼠树突状细胞成熟过程及其同种免疫刺激活性的影响(英文)

目的研究他克莫司对体外培养的大鼠树突状细胞(DC)成熟过程和同种刺激活性的影响。方法20只大鼠随机分为对照组、他克莫司、脂多糖(LPS)及LPS+他克莫司组,分别取骨髓细胞,加入粒细胞巨噬细胞集落刺激因子和白细胞介素4(IL-4)培养6d,收集贴壁细胞即DC。对照组不再加其他试剂,他克莫司组在开始培养时即加入他克莫司10μg·L-1,LPS组在细胞培养结束前18h加入LPS100μg·L-1,他克莫司+LPS组分别按上述要求加入他克莫司和LPS。用流式细胞术测定DC表面CD80和CD86的表达,ELISA方法检测其培养上清IL-12的浓度,用混合淋巴细胞培养法观察其对同种T细胞的刺激能力。结果与对照组比较,LPS刺激后,DC表面CD80和CD86表达明显增加,培养上清中IL-12浓度升高,其刺激同种T细胞的能力增强。与对照组和LPS组比较,他克莫司可使DC表面CD80和CD86表达明显降低,IL-12分泌减少,刺激同种T细胞的能力减弱。结论他克莫司可抑制体外培养的DC成熟及其同种刺激活性。     

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.     

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。     

胎儿来源的树突状细胞诱导抗膀胱癌效应的研究

目的：研究胎儿来源的树突状细胞（DC）体外诱导抗膀胱癌的特异性细胞免疫的效果.方法：从胎儿骨髓获得单个核细胞,经粒细胞-单核细胞集落刺激因子（GM-CSF）、IL-4和TNF-α诱导产生DC.利用50%～70%硫酸铵饱和沉淀法获取膀胱癌细胞系EJ含热休克蛋白（HSP）成分的细胞溶解物,以该抗原负载DC,激活胎脾细胞产生肿瘤特异性的细胞杀伤性T淋巴细胞（CTL）.利用IL-2刺激胎脾细胞产生LAK细胞.应用MTF法分别检测CTL和LAK细胞对EJ细胞的杀伤效应.结果：胎儿骨髓可诱导出功能成熟的DC,高表达CD1a、CD86、HLA-DR和CD83.负载EJ抗原的DC可诱导产生CD8＋CTL.其对EJ细胞的杀伤作用明显强于LAK细胞.结论：含HSP成分的肿瘤细胞溶解物负载胎儿来源的DC,体外可诱导出更强的特异性抗肿瘤免疫应答.     

基泰体外对慢性乙型肝炎患者树突状细胞功能影响的初步研究

目的:探讨基泰对慢性乙型肝炎(简称慢乙肝)患者(chronic hepatitis B,CHB)树突状细胞(dentritic cell,DC)生物学性状的影响.方法:分离慢乙肝患者外周血单核细胞,在含GM-CSF/IL-4及不同浓度的基泰培养条件下制备DC.观察DC形态学变化,流式细胞仪(FCM)测其细胞表型分子CD1a,CD83,CD80及HLA-DR的表达,MTT法测定DC刺激同种异体淋巴细胞增殖能力.结果:与对照组相比,实验组倒置显微镜观察显示出典型的DC形态.基泰(100 μg/mL)处理组的DC膜表面分子CD1a及CD80表达均增高(P<0.05);刺激同种异体淋巴细胞增殖能力(SI)增强(P<0.05).结论:基泰一定程度上可改善CHB的DC免疫学活性.     

肺癌抑制免疫功能的靶细胞：树突状细胞

目的：了解肿瘤微环境下肺癌细胞对DC前体细胞产生DC、功能的影响。方法：DC前体细胞为健康人外周血CD14细胞，加入GM－CSF和IL－4，37℃，5％CO2培养7－10天收获，肿瘤细胞株CRL－5815，CRL－5826加入DC前体细胞共培养，健康入外周血为正常对照，FITC或PE交链的单克隆抗体标记DC细胞，流式细胞仪分析检测CD14，CD86，CD80，CD1a阳性表达。Annexin V方法，流式细胞仪分析细胞凋亡，混合淋巴细胞反应（MLR）检测DC检测T细胞反应的能力。结果：肺癌细胞株可诱导DC形成的不同时期产生凋亡，对DC产生的早期（第1、2天）影响较晚期（第6，7天）更明显，肺癌细胞株CRL－5815和CRL－5826与DC共培养时明显抑制DC细胞表型CD80，CD86，CD40的表达，与肺癌细胞株共培养DC其刺激T细胞扩增的能力（MLR）显下降，结论：肺癌细胞株CRL－5815和CRL－5826与DC前体细胞共培养时可导致DC的凋亡显增加，同时DC细胞表面表型表达下降，刺激T细胞扩增的功能减低，提示肿瘤（肺癌细胞）可通过DC细胞数量减少和功能下降影响机体免疫功能。     

黄芪多糖促进人树突状细胞体外分化成熟的实验研究

目的：研究黄芪多糖（APS）对人树突状细胞（DCs）体外分化成熟及存活时间的影响。方法：健康志愿者外周血单个核细胞培养2 h获得贴壁的单核细胞,加入含rhGM-CSF（1000 U/mL）r、hIL-4（500 U/mL）的无血清培养基,实验组再加入3种浓度的APS（1μg/mL,10μg/mL,100μg/mL）,对照组加入等体积生理盐水,一组细胞体外培养7 d,收集悬浮细胞获得普通DCs（Mo-DCs）及APS诱导的DCs（APS-DCs）,流式细胞仪测定细胞免疫表型,同种异体混和淋巴细胞反应检测DCs的免疫激活功能,另外一组细胞体外连续培养14 d,光镜下观察细胞形态及存活时间。结果：APS能够显著促进DCs表面免疫分子HLA-DR、CD83、CD80、CD86、CD40、CD54的表达,APS-DCs较Mo-DCs具有更强的T细胞体外激活能力,并显著延长DCs的体外存活时间达2～3 d。结论：APS可以促进DCs的成熟和延长DCs的存活时间,从而发挥增强机体免疫力的功能。     

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.     

变形链球菌对EAhy926细胞TLR2 mRNA表达的影响

目的:观察变形链球菌细胞壁及培养上清对人血管内皮细胞EAhy926细胞TLR2 mRNA表达影响.方法:不同剂量的变形链球菌细胞壁或培养上清作用于EAhy926细胞,RT-PCR法检测EAhy926细胞TLR2 mRNA的表达.结果:变形链球菌细胞壁作用于EAhy926细胞后,TLR2 mRNA的表达量随着作用时间延长而逐渐增高,于16～24 h达到高峰,以后又逐渐下降(P<0.01);变形链球菌培养上清作用于EAhy926细胞后,TLR2 mRNA的表达量不随着作用时间和作用浓度而发生变化.结论:变形链球菌细胞壁可明显上调EAhy926细胞TLR2 mRNA的表达,并呈时间和剂量依赖性;变形链球菌培养上清不能刺激EAhy926细胞TLR2 mRNA的表达.     

1,25-二羟维生素D3对其诱导的树突状细胞上Toll样受体7表达的抑制作用

目的探讨Toll样受体7（TLR7）在1,25-二羟维生素D3（1,25-（OH）2D3）抑制树突状细胞（DC）成熟中可能的机制。方法体外扩增小鼠骨髓来源的DC,采用流式细胞术和混合淋巴细胞反应检测1,25-（OH）₂D₃对DC表型和功能的影响;利用RT-PCR半定量方法测定1,25-（OH）₂D₃对DC的TLR7表达的影响。结果 1,25-（OH）₂D₃处理后,DC表达CD86和MHCⅡ的荧光强度均明显降低;对T细胞的刺激能力较对照组明显减弱,差异有统计学意义（P<0.05）;与对照组比较,1,25-（OH）₂D₃可以明显下调TLR7的表达（P<0.05）。结论 1,25-（OH）₂D₃可能通过影响TLR7的表达来抑制DC细胞的成熟,从而使DC具有耐受性特征。     

A novel role for RGMa in modulation of bone marrow-derived dendritic cells maturation induced by lipopolysaccharide

Repulsive guidance molecule a (RGMa) is known to mediate immune responses and has been indicated to modulates T cell activation and autoimmune diseases by dendritic cells (DCs), which hints its significant function in the latter cells. The aim of our study, therefore, was to evaluate the function of RGMa in DC maturation. We found that small interfering RNA (siRNA) successfully silenced the expression of RGMa in DCs. Even after LPS stimulation, RGMa-silenced DCs displayed an immature morphology, characterized by small, round cells with a few cell processes and organelles, and many pinocytotic vesicles. In the presence of LPS, RGMa siRNA transfection markedly reduced levels of CD80, CD86, CD40, and MHC II expression, as well as the secretion of IL-12p70 and TNF-α. With LPS treatment, RGMa siRNA-transfected DCs also showed increased levels of IL-10 and endocytosis. Moreover, in the presence of LPS, RGMa siRNA-transfected DCs displayed a low ability to induce T cell proliferation and differentiation, compared with negative control (NTi)-transfected or control DCs (p < 0.05 for both). We conclude that after LPS stimulation, RGMa siRNA-transfected DCs show immunoregulatory and tolerogenic characteristics, which provides new insights into the immune system.     

Promoting effect of polysaccharide isolated from Mori fructus on dendritic cell maturation

Maturation of dendritic cells (DCs) is usually attenuated in the tumor microenvironment, which is an important immunological problem in DC-based immunotherapy of cancer. In this study, we report the effect of a Mori fructus polysaccharide (MFP) on DC maturation. MFP was treated to DCs generated from mouse BM cells. MFP induced phenotypic maturation of DCs, as proven by the increased expression of CD40, CD80/86, and MHC-I/II molecules. MFP induced functional maturation of DCs, in that MFP increased the expression of IL-12, IL-1β, TNF-α, and IFN-β, decreased antigen capture capacity, and enhanced allogenic T cell stimulation. MFP efficiently induced maturation of DCs from C3H/HeN mice having normal toll-like receptor4 (TLR4), but not DCs from C3H/HeJ mice having mutated TLR4, suggesting that TLR4 might be one of the membrane receptors of MFP. As a mechanism of action, MFP increased phosphorylation of mitogen-activated protein kinase (MAPKs), and nuclear translocation of NF-κB p65 subunit, which were important signal molecules downstream from TLR4. These data suggest that MFP induces DC maturation through TLR4 and MFP can be used as an adjuvant in DC-based cancer immunotherapy.     

IL-12 suppression,enhanced endocytosis and up-regulation of MHC-II and CD80 in dendritic cells during experimental endotoxin tolerance

Aim:

The aim of this study was to investigate endocytosis, MHC-II expression and co-stimulatory molecule expression, as well as interleukin-12 (IL-12) production, in bone marrow dendritic cells (DCs) derived from endotoxin tolerant mice.

Methods:

Endotoxin tolerance was induced in C57BL/10J mice through four consecutive daily intraperitoneal injections of 1.0 mg/kg of 055:B5 Escherichia coli lipopolysaccharide (LPS). Bone marrow DCs were isolated in the presence of GM-CSF and IL-4 and purified by anti-CD11c Micro beads. FITC–dextran uptake by DCs was tested by flow cytometric analysis and the percentage of dextran-containing cells was calculated using a fluorescence microscope. The expression of surface MHC-II, CD40, CD80, and CD86 was also detected by flow cytometric analysis. An ELISA was used for the measurement of IL-12 production by DCs with or without LPS stimulation.

Results:

Endotoxin tolerance was successfully induced in C57BL/10J mice, evidenced by an attenuated elevation of systemic TNF-α. DCs from endotoxin tolerant mice possessed enhanced dextran endocytosis ability. The expression of surface MHC-II and CD80 was higher in DCs from endotoxin tolerant mice than in DCs from control mice, whereas the expression of CD40 and CD86 was not altered. Compared with DCs from normal control mice, DCs from endotoxin tolerant mice produced less IL-12 after subsequent in vitro stimulation with LPS.

Conclusion:

These data suggest enhanced endocytosis, selective up-regulation of MHC-II and CD80 and IL-12 suppression in DCs during in vivo induction of endotoxin tolerance.     

Maturation of dendritic cells induced by Candida beta-D-glucan

We investigated whether Candida beta-D-glucan (CSBG) alters the maturation of dendritic cells (DCs). DC phenotypes were analyzed using FACScan. The expression of surface molecules, including major histocompatibility complex (MHC) classes I and II, as well as CD80 and CD86, increased on DCs that were stimulated with lipopolysaccaride (DCs/LPS), in comparison with unstimulated bone marrow-derived DCs (BM-DCs). Furthermore, the level of surface molecule expression on DCs stimulated with CSBG (DCs/CSBG) was between that of DCs and DCs/LPS. Phagocytosis was assessed by the uptake of FITC-dextran. There were no differences in the uptake of dextran among DCs/LPS and DCs/CSBG. The ability of BM-DCs to uptake dextran was higher than that of DCs/LPS and DCs/CSBG. We analyzed the concentration of IL-12 secreted by DCs using ELISA. BM-DCs secreted a low concentration of IL-12, while DCs/LPS and DCs/CSBG secreted higher levels of IL-12 than BM-DCs. There were no remarkable differences in the concentrations of IL-12 produced by DCs/LPS and DCs/CSBG. This data suggests that CSBG may augment DC maturation.     

青心酮对小鼠动脉粥样硬化斑块中主要细胞TLR4表达的影响

目的:研究青心酮对小鼠动脉粥样硬化(AS)斑块中主要细胞(平滑肌细胞、内皮细胞、巨噬细胞)Toll样受体4(TLR4)表达的影响。方法:取小鼠胸主动脉,进行平滑肌细胞、内皮细胞的原代培养,RAW264.7细胞株传代培养。实验分5组,即对照、脂多糖(LPS)、辛伐他汀和青心酮高、低剂量组。应用RT-PCR及Western-blot方法检测TLR4mRNA表达及蛋白含量。结果:青心酮高剂量组血管平滑肌细胞、内皮细胞、巨噬细胞TLR4mRNA和蛋白含量显著低于LPS组(P<0.01)。结论:青心酮可下调LPS活化的小鼠平滑肌细胞、内皮细胞、巨噬细胞TLR4mRNA和蛋白的表达,其机制可能与通过TLR4途径发挥抗炎作用有关。     

A novel polysaccharide from the seeds of Plantago asiatica L. induces dendritic cells maturation through toll-like receptor 4

In this study, we investigated the effect of a polysaccharide purified from the seeds of Plantago asiatica L. (PLP-2) on the phenotypic and functional maturation of murine bone marrow-derived dendritic cells (DCs) and relevant mechanisms. The results showed that PLP-2 increased the expression of maturation markers major histocompatibility complex II, CD86, CD80, and CD40 on DCs. Consistent with the changes in the phenotypic markers, functional assay for DCs maturation showed that PLP-2 decreased DCs endocytosis and increased intracellular interleukin (IL)-12 levels and allostimulatory activity. Furthermore, using a syngeneic T cell activation model, we found that PLP-2 treated DCs presented ovalbumin antigen to T cells more efficiently as demonstrated by increased T cell proliferation. In addition, the effects of PLP-2 on DCs were significantly impaired by treating the cells with anti-TLR4 antibody prior to PLP-2 treatment, implying direct interaction between PLP-2 and TLR4 on cell surface. These results suggested that PLP-2 may induce DCs maturation through TLR4. Our results may have important implications for our understanding on the molecular mechanisms of immunopotentiating action of the polysaccharides from plants.