启膈散对食管癌EC9706细胞株抑制树突状细胞成熟的影响

目的观察启膈散对食管癌细胞干预树突状细胞成熟的影响。方法制备条件培养基,用Fieoll密度梯度离心法分离外周血单个核细胞,体外诱导培养树突状细胞(Dendritic Cells,DCs),流式细胞术检测DCs表面标志物,MTT法检测淋巴细胞细胞增殖;LDH释放法检测细胞毒T淋巴细胞(cytotoxic T lymphocyte,CTL)杀伤力;ELISA检测细胞因子分泌,Western blot检测STAT3蛋白表达。结果食管癌EC9706细胞条件培养基可以减少外周血单核细胞诱导的树突状细胞表达CD80、CD86、CD1a,抑制DCs刺激淋巴细胞增殖和减弱CTL的杀伤力,减少IL~(-1)2分泌,增加STAT3蛋白表达和磷酸化;与肿瘤条件培养基相比,启膈散条件培养基组DCs CD80、CD86、CD1a增加,淋巴细胞的增殖能力、CTL杀伤作用增强,IL~(-1)2分泌增加,STAT3蛋白表达和磷酸化减少。结论启膈散可以通过STAT3信号通路减弱食管癌细胞上清对树突状细胞成熟的影响。     

树突状细胞的培养与鉴定

目的研究利用磁珠分离方法获取单核细胞,用细胞因子GM—CSF和IL-4联合诱导使之分化为树突状细胞（dendritic cells,DCs）的方法,并对培养的细胞从形态,表型,功能等三个方面进行鉴定,从而探索出一套较成熟的DCs的培养方法。方法通过密度梯度离心的方法获取白细胞悬液中的白膜层,然后通过磁珠分离的方法,收集高纯度的单核细胞。在细胞因子GM—CSF和IL-4的刺激下,将细胞培养至7天左右,收集细胞进行流式分析,并进行淋巴细胞增殖实验,鉴定细胞是否为树突状细胞。结果在倒置显微镜下观察,细胞培养第7天,大部分细胞呈单个悬浮,并有明显的毛刺样突起。流式分析发现细胞高表达DC表面特异性抗原CD80,CD86,HIA—DR,并且不再表达单核细胞表面抗原CD14,细胞纯度约为93．12％。淋巴细胞增殖实验显示DCs可明显刺激初始型淋巴细胞增殖。结论通过磁珠分离的方法获得单核细胞并利用GM-CSF和IL-4细胞因子诱导可成功培养出纯度高的树突状细胞。     

青藤碱对类风湿关节炎病人树突状细胞免疫活性的影响

 目的观察中药单体青藤碱对体外培养类风湿关节炎树突状细胞免疫活性的影响。方法分离12例类风湿关节炎病人外周血单核细胞,分为青藤碱高、中、低剂量组和空白对照组。采用GM-CSF,IL-4进行刺激分化,于分化第4天开始给予干预,于培养第8天收获细胞。混合淋巴细胞反应检测树突状细胞对同种异体T细胞的刺激作用,酶联免疫吸附法检测树突状细胞培养上清中IL-12的含量,流式细胞技术检测树突状细胞表型的改变。结果与对照组相比,在不同混合比例下青藤碱可抑制树突状细胞对同种异体T细胞的刺激能力,青藤碱可抑制树突状细胞IL-12的分泌及其表面分子CD40,CD80,CD83,CD86和HLA-DR的表达。结论青藤碱可能通过抑制树突状细胞激活T细胞的能力,而达到治疗类风湿关节炎的作用。     

华蟾素对慢性乙型肝炎患者外周血来源树突状细胞的影响

目的:研究华蟾素对慢性乙肝患者外周血来源的树突状细胞(dendritic cells,DCs)形态、表型和功能的影响,以了解华蟾素提高慢乙肝患者机体免疫力的作用机制。方法:实验研究分3组:正常对照组、慢乙肝组和慢乙肝华蟾素组。分离慢性乙肝患者和正常人外周血单核细胞(Monocytes,Mo),以GM-CSF和IL-4联合诱导培养生成DCs,慢乙肝华蟾素组于DCs培养48小时后加入华蟾素。各组DCs均于培养的第7天以相差显微镜观察和拍摄细胞形态;用流式细胞仪检测表面分子CD40、HLA-DR、CD80、CD86的表达水平;用CCK8法检测DCs刺激同种异体淋巴细胞增殖的能力;用ELISA法检测DCs培养液上清中IL-12的水平。结果:与正常人相比,慢乙肝组患者外周血来源的DCs细胞形态相对不成熟,表面分子表达水平低,刺激同种异体淋巴细胞增殖能力和IL-12分泌能力较弱;加入华蟾素培养的慢乙肝患者DCs与慢乙肝组比较,细胞形态相对成熟,表面分子CD40、CD80和CD86表达水平增高,刺激同种异体淋巴细胞增殖能力和IL-12分泌能力增强。结论:华蟾素能促进慢性乙肝患者外周血来源DCs的发育成熟,改善其功能。     

当归多糖对树突状细胞成熟和功能的影响

目的:探讨当归多糖对外周血单个核细胞(PBMC)来源的树突状细胞(DC)体外分化、成熟和功能的影响。方法:以外周血单个核细胞为来源,联合应用 GM-CSF、IL-4及 TNF-α,在体外诱导培养 DC。采用 MTT 法测定 DC 诱导的混合淋巴细胞反应分析 APS-3对 DC 刺激淋巴细胞增殖功能的影响;采用流式细胞术检测细胞表面标志 CD1a,HLA-DR,CD86的表达,来评价 APS-3对 DC 分化的作用;RT-PCR 技术研究 DC IL-12p40mRNA 的表达,探讨 APS-3对 DC 分泌 Th1型细胞因子 IL-12的影响。结果:人 PBMC 在体外可诱导培养出具有典型形态、表型及免疫活性的 DC。采用不同浓度APS-3(0.1、0.3、1.0μg/mL)处理培养的 DC 表面标志 CD1a、CD86、HLA-DR 及 IL-12mRNA 表达量均呈上升趋势。经不同浓度 APS-3处理的 DC 诱导的 MLR,在小剂量范围(0.01-1μg/mL)是剂量依赖性的增强作用,在高剂量范围(30-100μg/mL)表现为抑制效应;常规培养8天生成的 DC 与淋巴细胞共培养后,加入不同浓度 APS...     

重组人p53腺病毒转染急性早幼粒细胞白血病源性树突状细胞的免疫效应研究

目的研究重组人p53腺病毒(rAd-p53)转染急性早幼粒细胞白血病(APL)源性树突状细胞(DC)的免疫效应。方法采集APL初治患者骨髓单个核细胞在完全培养基中培养并以r-Ad-p53/Lac Z(腺病毒空载体)处理。Western blot鉴定p53蛋白的表达,流式细胞仪检测DC免疫表型、酶联免疫吸附法(ELISA)进行上清夜IL-12水平测定,MTT法观察rAd-p53-APL-DC刺激自体T淋巴细胞增殖能力(MLR)。结果转染rAd-p53或Lac Z可明显上调APL-DC表面CD80、CD86、CD83、CD1a、CD40和HLA-DR表达;转染rAd-p53后72小时rAd-p53-APL-DC上清液中IL-12分泌水平明显增加;刺激自体淋巴细胞增殖的能力随rAd-p53-APL-DC与淋巴细胞比例的增加而升高(P0.05).结论利用rAd-p53转染急性早幼粒细胞白血病细胞来源树突状细胞(APL-DC),可有效刺激APL-DC的成熟,其介导的MLR明显强于非rAd-p53组所诱导的DC。     

黄芪多糖对小鼠树突状细胞前体的体内诱导研究

目的探讨黄芪多糖(astragalus polysaccharide,APS)在体内是否具有诱导小鼠髓源性树突状细胞(dendritic cell,DC)分化和成熟作用,以阐释APS药理作用机制。方法分别设对照组及APS100、200mg/kg剂量组,腹腔注射,体内诱导小鼠髓源性DC前体和未成熟DC,称量脾重,ELISA检测血清中GM-CSF表达量,倒置显微镜观察细胞生长状态,电镜检测细胞形态,流式细胞仪检测CD11c、MHC-Ⅱ类分子及协同刺激分子CD80、CD86表达水平。结果APS注射7d后,APS100、200mg/kg组小鼠脾脏重量均较对照组有明显增加(P<0.01),骨髓细胞体外分离并经rmGM-CSF、rmIL-4诱导培养后,流式细胞检测结果显示:APS100、200mg/kg组CD11c、MHC-Ⅱ类分子表达水平高于对照组(P<0.05),但CD80、CD86表达量较对照组无明显差异;血清中GM-CSF表达量,结果与对照组无明显差异。结论APS能诱导小鼠髓源性DC前体分化为DC,并可能有促进DC成熟的作用,但此作用并非通过刺激机体GM-CSF细胞因子的分泌而发挥作用。     

苯甲酰乌头原碱对HBV-ACLF阳黄证和阴黄证患者外周血树突细胞功能的影响

目的：研究苯甲酰乌头原碱（Benzoylaconine, BAC）对HBV相关慢加急性肝衰竭（HBV related acute-on-chronic liver failure, HBV-ACLF）阳黄证和阴黄证患者外周血树突状细胞（dendritic cells, DCs）功能的影响。方法：体外诱导培养HBV-ACLF阳黄证与阴黄证患者DCs,CCK-8法检测BAC安全用药范围，流式检测BAC干预HBV-ACLF阳黄证与阴黄证患者DCs表面CD80、CD86、CD83和HLA-DR表达率变化情况，ELISA法检测DCs分泌IL-12水平。结果：与健康对照组比较，阳黄证、阴黄证空白组DCs表面CD80、CD86、CD83、HLA-DR表达降低（P <0.01）,IL-12分泌减少（P <0.01）；与阳黄证、阴黄证空白组比较，阳黄证、阴黄证BAC干预组DCs表面CD80、CD86、CD83、HLA-DR表达升高，IL-12分泌增多（P <0.01）。结论：BAC可以促进HBV-ACLF阳黄证和阴黄证患者DCs功能恢复，间接促进T细胞活化。     

黄芪多糖诱导脐血单核细胞分化为树突状细胞的鉴定分析

目的:观察黄芪多糖对脐血单核细胞分化为树突状细胞的诱导作用并对其进行分析鉴定.方法:无菌条件下采集脐血,密度梯度离心法获得脐血单核细胞,分为3组,即实验组、阴性对照组、阳性对照组;培养过程中用倒置光学显微镜和透射电镜观察细胞形态;收集部分培养第12天的细胞利用流式细胞仪检测各组细胞表面CD1a、CD80、CD83和CD86的表达.结果:在培养72小时后实验组和阳性对照组细胞形态开始变化,随着培养时间的延长,树突状结构更加明显,第12天细胞呈典型的树突状细胞形态;阴性对照组细胞生长缓慢,培养至第12天细胞呈梭形巨噬细胞形态.培养至10天的实验组细胞透射电镜下呈典型的树突状细胞形态.培养12天实验组、阳性对照组细胞分别高表达DCs特异性抗原CD1a、CD80、CD83和CD86,与阴性对照组对应比较差异均有显著性(P＜0.01);实验组与阳性对照组之间比较无统计学意义(P＞0.05).结论:黄芪多糖体外可诱导脐血单核细胞(DCs前体细胞)定向分化为树突状细胞(DCs).     

HepG2肝癌细胞外泌体对人树突状细胞成熟与功能的影响

目的:观察HepG2肝癌细胞外泌体对人树突状细胞(Dendritic cells,DC)成熟和功能的影响。方法:采用透射电镜、Nano FCM和Western Blot鉴定和测定外泌体的形态、粒径分布及特征性蛋白的表达,用细胞因子GM-CSF(20 ng/ml)、IL-4(10 ng/ml)、IFN-β(105U/ml)和LPS(10 ng/ml)诱导刺激DC成熟,用流式细胞术检测DC成熟相关表面分子CD80、CD83、CD86、HLA-DR的表达,ELISA检测培养上清液中细胞因子IL-12的水平,CCK-8法检测DC刺激同种异体淋巴细胞增殖的能力。结果:HepG2、MIHA来源外泌体表达CD63、Alix,同时不表达细胞色素C(Cytochrome C);在HepG2肝癌细胞外泌体刺激下,DC的CD80、CD83阳性表达率较仅加入LPS的对照组明显降低(P<0.05),而CD86、HLA-DR表达较对照组无明显差异;ELISA测定结果显示,200μg/ml HepG2 EXO干预后的DC较对照组分泌IL-12的量明显减少(P<0.05);CCK-8检测显示,200μg/ml HepG2EXO干预的DC较对照组刺激异体淋巴细胞增殖的能力明显降低(P<0.05)。结论:HepG2肝癌细胞外泌体对DC表面成熟相关分子表达、细胞因子的分泌、刺激异体T淋巴细胞增殖的能力均有一定的抑制作用。     

大黄素体外抑制人树突状细胞分化及成熟作用的研究

目的 探讨大黄素体外作用树突状细胞（dendritic cell, DC）后对人DC分化、成熟及免疫功能的影响。     

参附注射液联合细胞因子对人慢性髓性白血病细胞来源树突状细胞诱导生成的影响

目的：观察不同浓度参附注射液（SFI）联合细胞因子（GM-CSF/IL-4）对慢性髓性白血病（CML）来源树突状细胞（DC）成熟与功能的影响。方法：采用CML患者的单个核细胞（MNC）,体外诱导扩增DC,在培养体系中分别添加不同浓度的SFI（终浓度分别为：低浓度9.38 mg/mL;中浓度37.50、75 mg/mL;高浓度150、300 mg/mL生药浓度）、GM-CSF/IL-4及不同浓度SFI联合细胞因子,通过观察细胞形态、细胞表面分子表达水平、细胞功能的变化,对所获得的细胞进行鉴定。结果：体外单用SFI不能诱导CML细胞分化为DC;细胞因子诱导的CML-DC存在表型及功能障碍;而在含有细胞因子的培养体系中添加SFI,对CML-DC有促进成熟和抑制增殖的双向调节作用,添加高浓度SFI可抑制DC的生长甚至导致细胞死亡,添加中浓度SFI能获得较多成熟CML-DC,可提高其细胞表面共刺激分子的表达水平,并可改善其刺激淋巴细胞增殖的能力及IL-12的含量。结论：适当浓度的SFI可影响CML-DC的分化和成熟,并可改变DC细胞表面免疫分子的表达,增强其功能;SFI最佳诱导浓度为75 mg/mL,且75 mg/mL SFI与GM-CSF/IL-4联合应用有协同作用。     

Armillariella mellea induces maturation of human dendritic cells without induction of cytokine expression

ETHNOPHARMACOLOGICAL RELEVANCE: Armillariella mellea, an edible and medicinal mushroom possessing immuno-modulating potential, has been frequently used for the treatment of infectious diseases or cancers. AIM OF THE STUDY: In order to elucidate immune-regulatory mechanisms of Armillariella mellea, we investigated the effect of water-soluble components from Armillariella mellea (AME) on the regulation of human dendritic cell (DC) maturation and activation. MATERIALS AND METHODS: Immature DCs (iDCs) were prepared by differentiating human peripheral blood CD14-positive cells with GM-CSF and IL-4. Then, iDCs were treated with AME at 2-20mug/ml for 48h and subjected to flow cytometry to analyze the expression of DC markers. Dextran-FITC uptake assay and enzyme-linked immunosorbent assay were performed to examine the endocytic capacity of AME-stimulated DC and their production of cytokines, respectively. RESULTS: iDCs stimulated with AME showed representative features during DC maturation such as up-regulated expression of CD80, CD83, CD86, both MHC class I and II molecules, and CD205, with a simultaneous decrease in the expression of CD206 and the endocytic capacity. Interestingly, AME was not able to induce the production of TNF-alpha, IL-12p40, or IL-10, whereas lipopolysaccharides induced a substantial increase of all of the cytokines. CONCLUSION: Armillariella mellea induces maturation of human DCs through a unique mechanism without inducing cytokine expression.     

Panax quinquefolium saponins inhibited immune maturation of human monocyte-derived dendritic cells via blocking nuclear factor-κB pathway

Ethnopharmacological relevance

Panax quinquefolium saponins (PQS), a water-soluble antioxidant extracted from a natural herb, radix panacis quinquefolii (American Ginseng), has yielded encouraging results in the treatment of atherosclerotic diseases. However, the underlying mechanisms remain unclear. Here, we tested the hypothesis that the anti-atherosclerotic effect of PQS might be mediated by suppressing human monocyte-derived dendritic cells (DCs) maturation.

Materials and methods

DCs were derived by incubating purified human monocytes with granulocyte macrophage colony stimulating factor (GM-CSF) and IL-4. DCs were pre-incubated with or without PQS and stimulated by oxidized low density lipoprotein (ox-LDL). Expression of DCs membrane molecules (CD40, CD86, CD1a, HLA-DR) and endocytotic ability were analyzed by FACS, cytokines (IL-12 and TNF-α) were measured by ELISA. Nuclear factor (NF)-κB signaling pathway was determined by Western blotting, and RT-PCR. NF-κB activation was quantified by ELISA.

Results

PQS reduced ox-LDL induced immunophenotypic expressions (CD40, CD1a, CD86, and HLA-DR) and cytokine secretions (IL-12 and TNF-α), and improved endocytotic ability of DCs. These above phenomena were accompanied by decreased protein expression and binding activity of nuclear localized c-Rel subunit.

Conclusions

Our study suggested that PQS inhibited ox-LDL induced immune maturation of DCs in vitro, which might be in part mediated by NF-κB signal transduction pathway.     

In vitro immunomodulatory activities of a newly concocted traditional Chinese medicine formula: VI-28

Previous studies have suggested that Vigconic VI-28, an anti-aging traditional Chinese medicine (TCM) formula containing Radix Ginseng and Cornu Cervi Pantotrichum, possesses immunological efficacy. This in vitro study further investigated the immunomodulatory effects of the hot water extracts of VI-28. The study included (1) colorimetric 5-bromo-2'-deoxy-uridine proliferation ELISA for estimating mitogenicity in human peripheral blood mononuclear cells (PBMC), (2) immunofluorescence staining for measuring the expression of IL-2 receptor alpha (CD25) on lymphocytes, (3) cytometric bead array (CBA) for quantifying cytokine liberation from PBMC, and (4) intracellular immunophenotyping for macrophage phagocytosis and hydrogen peroxide (H(2)O(2)) production from monocytes. The results demonstrated that VI-28 (1) could dose-dependently inhibited the proliferation of unstimulated and lipopolysaccharide-activated PBMC but enhanced the proliferation of phytohemagglutinin-activated PBMC at concentrations of <1 mg/mL, (2) significantly augmented the expression of CD25 on lymphocytes at concentrations of 0.4 mg/mL or above (p < 0.05), (3) dose dependently (0.1-1.0 mg/mL) activated macrophage phagocytosis and monocyte synthesis of H(2)O(2) and (4) significantly increased the production of cytokines IL-8, IL-10, IL-12 and IL-1beta at various concentrations of VI-28 (p < 0.05). The results suggest that VI-28 is a potential immunomodulator which probably acts through the activation of lymphocytes and monocytes.     

复方血尿停经肝微粒体代谢后对肾小球系膜细胞的影响

目的探讨复方血尿停治疗以系膜增生为主要病理改变的肾小球疾病的作用机制。方法将复方血尿停与大鼠肝微粒体共同孵育后加入到肾小球系膜细胞(GMC)体外培养体系中,MTT法观察细胞增殖情况,放射免疫法测定白细胞介素-6(IL-6)、内皮素-1(ET-1)水平,速率法检测乳酸脱氢酶(LDH)的含量。结果经肝微粒体代谢后,复方血尿停2、4、8mg/mL均抑制GMC的增殖及IL-6、ET-1的产生,且呈剂量依赖方式。结论复方血尿停能抑制IL-6、ET-1的分泌和GMC的增殖,此作用可能是其治疗系膜增生性肾小球疾病的作用机制之一。     

Brazilian red propolis exerts a cytotoxic action against prostate cancer cells and upregulates human monocyte functions

Different propolis samples can be obtained in Brazil, such as green, brown and red. Studies related to Brazilian red propolis (BRP) have increased in the last few years, so the aim of this study was to investigate its effects on the prostate cell lines LNCaP and PC-3 and on human monocytes. BRP chemical composition was analyzed by HPLC-DAD, the viability of monocyte and cancer cell by MTT assay. Cytokine production (TNF-α, IL-1β, IL-6, IL-10) by monocytes was quantitated by ELISA, the expression of cell markers (TLR-2, TLR-4, HLA-DR, CD80) and reactive oxygen species by flow cytometry. The candidacidal activity and the effects of supernatant of treated monocytes on tumor cells were assessed. BRP affected LNCaP viability after 48 and 72 h, while PC-3 cells were more resistant over time. BRP upregulated CD80 and HLA-DR expression, and stimulated TNF-α, IL-6 and IL-10 production. BRP enhanced the fungicidal activity of monocytes, displayed an antioxidant action and the supernatant of BRP-treated monocytes diminished LNCaP viability. In the search for new immunomodulatory and antitumoral agents, BRP exerted a selective cytotoxic activity on prostate cancer cells and an immunomodulatory action, suggesting its potential for clinical trials with oncological patients and for the discovery of new immunomodulatory and antitumor drugs.