芍药苷对树突状细胞表型成熟及抗炎因子分泌的影响

分离培养小鼠骨髓树突状细胞,加入诱导变应性接触性皮炎变应原(DNCB)刺激DC,通过体外实验观察芍药苷(PF)对树突状细胞(DC)的表面分子MHC II、CD40、CD80、CD86及促炎因子IL-12p70和抗炎因子IL-10分泌的影响.PF抑制由DNCB引起DC的MHC II高表达和刺激分子CD80、CD86、CD40的表达上调,且呈剂量依赖型;同时伴随抑制由DNCB诱导的促炎因子IL-12p70的产生(P<0.05),促进抗炎症因子IL-10的分泌(P<0.05),但对TGF-β1的分泌没有影响(P>0.05).芍药苷通过抑制DNCB诱导的DC表型成熟及炎性细胞因子产生,发挥了免疫抑制作用,在防治变应性接触性皮炎等免疫相关疾病中发挥了重要作用.     

复发性外阴阴道念珠菌病患者树突细胞Dectin-1受体的功能研究

目的 对比复发性外阴阴道念珠菌病（RVVC）患者和健康女性树突细胞（DC）表面Dectin-1受体信号传导及功能的差异,分析患者病情反复发作的可能原因。 方法 提取1例RVVC患者和1例健康女性的单核细胞,诱导分化为DC。DC与白念珠菌共培养后,流式细胞仪测定DC表面CD83、CD86和CD80表达水平,分析细胞的成熟率;Western印迹法测定DC的Dectin-1、酪氨酸激酶（Syk）和CARD9蛋白的表达;ELISA法测定DC分泌白细胞介素23（IL-23）、肿瘤坏死因子α（TNF-α）和IL-12水平。 结果 与健康人DC相比,与白念珠菌共培养24 h后,该RVVC患者DC表面CD83、CD86和CD80的表达活化不明显。与健康人DC相比,与白念珠菌共培养2 h后,RVVC患者DC表达的Dectin-1没有显著性差异,但是磷酸化Syk和CARD9活化障碍。与健康人DC相比,与白念珠菌共培养6 h后,RVVC患者DC分泌的IL-23、TNF-α和IL-12升高也不明显。抗人Dectin-1抗体对RVVC患者DC的Syk依赖的信号传导通路和上述细胞因子的分泌都没有进一步抑制作用。 结论 该RVVC患者DC的Dectin-1受体信号传导通路障碍,导致DC成熟率降低,分泌的IL-23、TNF-α和IL-12降低,使得宿主黏膜抗念珠菌感染的天然免疫功能缺陷。     

HPV-11 E7重组腺病毒体外转染树突细胞对其功能的影响

目的 研究腺病毒载体介导HPV-11 E7基因转染树突细胞(DC)对其表型和功能的影响。方法 用最佳感染滴度(MOI)重组腺病毒pAD-E7转染成熟DC,流式细胞仪检测转染前后DC表型变化,并用~3H-TdR掺入法检测转染前后DC刺激同种淋巴细胞增殖的能力及ELISA检测DC、T细胞共同培养上清中细胞因子的水平。结果 MOI 100为pAD-E7转染DC最佳滴度,pAD-E7转染成熟DC前后对细胞表面的特征性表型CD1a、CD83及CD40、HLA-DR无影响,转染后的DC仍具有较强的刺激同种异体淋巴细胞增殖的能力,能刺激T细胞分泌大量IFN-γ、IL-12。结论 pAD-E7转染成熟DC对其功能无明显影响,转染后的DC与T细胞共孵育能诱导T细胞向Th1细胞极化。     

黑素瘤患者树突状细胞体外快速培养成熟和临床应用研究

目的：建立从黑素瘤患者外周血体外快速稳定地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患者外周血中分离出的单个核细胞（PBMC）,再用贴壁法获取单个核细胞后加入粒-巨噬细胞集落刺激因子（GM—CSF）和白介素（IL）-4,随后分4组：第1和第2组分别在24h时加入多聚次黄嘌呤胞嘧啶核苷酸[Poly（I：C）]、肿瘤坏死因子（TNF）-α,继续培养24h,在48h时收集DC;第3和第4组分别在36h时加入Poly（I：C）、TNF—α。继续培养36h,在72h时收获DC。显微镜下观察DC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：经GM—CSF、IL-4、Poly（I：C）诱导培养72h获得大量成熟DC,形态学观察可见典型特征,荧光激活细胞分离器（FACS）检测表明。第3组DC高表达CD83、CD80、CD86和CD40;混合淋巴细胞反应（MLR）表明,第3组获得的DC具有较强的刺激同种淋巴细胞增殖能力。获取的成熟DC注射于患者浅表淋巴结后未出现明显不良反应。结论：Poly（I：C）体外诱导人外周血单个核细胞来源Dc成熟的方法具有快速、稳定、经济、安全的特点。培养后DC回输患者体内未出现明显不良反应。     

梅毒患者外周血树突细胞亚型及其表达不同细胞因子的检测

目的 探讨梅毒患者外周血树突细胞（DC）各亚型及表达不同细胞因子T细胞亚群的频数在梅毒发病机制中的意义。方法 流式细胞仪测定20例梅毒患者治疗前、治疗后和15例正常人外周血DC各亚型及表达不同细胞因子（包括IFN-γ、IL-2、IL-4、IL-10、IL-12）CD4+、CD8+ T细胞亚群的频数。结果 20例梅毒患者外周血髓系DC亚型（CD11c＋）的表达，治疗前、后比较有显著升高（t = 3.339，P ＜ 0.01），治疗前与对照组比较显著升高（P ＜ 0.05）。梅毒患者外周淋巴系DC亚型（CD123＋）的表达，治疗前、治疗后、对照组比较其差异均无统计学意义（P ＞ 0.05）。梅毒患者外周血T细胞亚群胞质内细胞因子IFN-γ的表达，治疗前、后比较有显著升高（t =14.890，P ＜ 0.01），治疗前与对照组比较显著升高（P ＜ 0.01）；梅毒患者外周血T细胞亚群胞质内细胞因子IL-2、IL-4、IL-10、IL-12的表达，IL-2和IL-12治疗前、后比较有显著升高（t = 10.899，4.292，P均 ＜ 0.01），治疗前与对照组比较显著升高（P ＜ 0.05），IL-4和IL-10治疗前与对照组比较显著降低（P ＜ 0.01）。梅毒患者外周血CD11c＋、CD123＋的表达与IFN-γ、IL-12的百分比不存在相关关系（P均 ＞ 0.05）。结论 IFN-γ、IL-2和IL-12可能在梅毒清除过程中发挥免疫调节作用，而IL-4、IL-10起免疫抑制作用。     

银杏叶提取物对系统性硬皮病患者外周血淋巴细胞增殖及功能的影响

目的 探讨银杏叶提取物对系统性硬皮病患者体外培养外周血淋巴细胞增殖及其分泌细胞因子功能的影响.方法 对系统性硬皮病患者和正常对照组的外周血淋巴细胞进行体外培养,分别加入不同浓度银杏叶提取物(EGB),60h后计算淋巴细胞的细胞增殖率并检测细胞因子IL-4和γ-IFN的分泌水平.结果 ①实验组,25g/L EGB对淋巴细胞增殖无明显促进作用(P=0.352),50g/L EGB对淋巴细胞增殖有明显的促进作用(P=0.000),100g/L EGB对淋巴细胞增殖促进作用较50g/L弱(P=0.044),与浓度有关;②实验组各浓度EGB干预后IL-4分泌量较空白对照无统计学差异;③实验组γ-IFN分泌量与干预的药物浓度有关,100g/L,50g/L EGB能促进γ-IFN分泌(P=0.000,P=0.019),25g/L EGB对γ-IFN分泌无明显影响(P=0.866);④不同浓度EGB对正常对照组体外培养外周血淋巴细胞增殖及IL-4,γ-IFN的分泌均无明显影响.结论 EGB体外能促进系统性硬皮病患者淋巴细胞增殖,并诱导γ-IFN分泌,且与浓度有关,提示EGB可能对系统性硬皮病有治疗作用.     

复方中药对银屑病患者外周血白介素12的表达

近年研究认为,银屑病是一种T细胞介导的自身免疫性疾病[1].本实验通过体外培养的寻常性银屑病患者外周血树突细胞(DC)IL-12的产生情况,研究复方中药对寻常性银屑病患者外周血Dc的IL-12 mRNA表达的影响.     

中药对培养小鼠CD8+细胞毒性T淋巴细胞的影响

【摘要】 目的 建立CD8+细胞毒性T淋巴细胞（CTL）体外增殖模型,筛选免疫抑制性中药。 方法 制备小鼠脾脏单细胞悬液,通过特异性抗体分离CD8+ T淋巴细胞,CD3/CD28抗体诱导淋巴细胞活化增殖,分别加入23种中药提取物共培养,通过四唑盐MTS法检测各中药对淋巴细胞增殖的抑制效果,对抑制作用最强的4种中药作12.5 ～ 400 mg/L的浓度梯度分析。酶联免疫斑点法（ELISPOT）检测这4种中药对CD3/CD28抗体诱导CD8+ T细胞分泌干扰素γ（IFN-γ）的作用。 结果 23种中药提取物中,14味中药对淋巴细胞增殖有不同程度抑制,抑制作用最强的前4味分别为黄连、黄芩、木香和姜黄,其对CD8+ T细胞增殖的50%抑制浓度（IC50）分别约为25、35、50和60 mg/L,100%抑制的最低浓度分别为200、100、200、200 mg/L。黄芩、木香和姜黄在100 mg/L的浓度下对CD3/CD28 抗体诱导CD8+ T细胞分泌IFN-γ均有明显抑制作用,而黄连对CD8+ T细胞分泌IFN-γ的抑制作用不明显。 结论 成功建立CD8+ CTL体外增殖模型,并筛选出对小鼠脾脏CD8+ T淋巴细胞增殖具有显著抑制作用的4味中药即黄连、黄芩、木香和姜黄。     

奥曲肽对系统性红斑狼疮患者体外培养单一核细胞增殖及分泌细胞因子的影响

目的 探讨奥曲肽对SLE患者体外培养单一核细胞增殖及其分泌细胞因子的影响。方法 对SLE患者和正常人对照的单一核细胞进行体外培养,用MTT法测定单一核细胞加入不同浓度奥曲肽后的细胞增殖率,计算不同浓度奥曲肽对单一核细胞的抑制率,同时以生长激素加奥曲肽进行干预,并测定细胞因子的分泌水平。结果 0.1 ng/mL奥曲肽对单一核细胞增殖无抑制作用,1 ng/mL奥曲肽开始有微弱的抑制作用,10 ng/mL奥曲肽有明显的抑制作用,且呈剂量依赖性。SLE患者外周血单一核细胞在体外经奥曲肽干预后,其IL-6、IL-10和IFN-γ分泌量较未处理组显著下降,生长激素加奥曲肽共同干预后的分泌量较生长激素干预的分泌量显著下降。结论 奥曲肽体外能抑制单一核细胞增殖,且能抑制生长激素诱导的细胞因子的分泌。     

干扰素γ诱导人黑素细胞CD40的表达

目的 探讨γ-干扰素(IFN-γ)体外诱导人黑素细胞表面CD40分子的表达及其意义.方法 常规分离培养人黑素细胞,流式细胞仪测定IFN-γ处理前后细胞表面CD40等免疫分子表达;混合淋巴细胞反应评价黑素细胞对同种异体T淋巴细胞的刺激能力;ELISA检测培养上清液IL-8、IL-10、IL-12的浓度.结果 体外培养的人黑素细胞表面表达少量CD40分子;不同浓度的IFN-γ处理黑素细胞24h、48h、72h后,能显着促进其CD40的上调(P<0.01),且24h处理组的黑素细胞CD40的表达量和IFN-γ的浓度呈直线相关.经IFN-γ诱导后的黑素细胞形态有所变化,刺激同种异体淋巴细胞的能力也显着增加,300IU/mLIFN-γ处理的黑素细胞72h刺激指数(SI)可达到峰值.黑素细胞经IFN-γ作用后,培养上清液中IL-12水平明显增加(P<0.05),而IL-8、IL-10的浓度无变化(P>0.05).经IFN-γ预处理的黑素细胞经SCD40L配基化后,能显着上调CD80、细胞间粘附分子1(P<0.01),且这种作用能被特异性的CD40L的单克隆抗体所阻断.结论 IFN-γ体外能够诱导黑素细胞功能性地表达CD40分子及增加对淋巴细胞的刺激能力,这对于认识黑素细胞在细胞免疫应答中的作用具有重要的意义,CD40分子上调后,黑素细胞可能不经过CD4⁺细胞而直接刺激活化CD8⁺的杀伤性T细胞(CTL).     

Protective effect of epigallocatechin gallate on the immune function of dendritic cells after ultraviolet B irradiation

Aim  To investigate the protective effect of epigallocatechin gallate (EGCG) on the immune function of dendritic cells (DCs) after ultraviolet B irradiation (UVB) and its underlying mechanisms.
Methods  The monocytes were isolated from peripheral blood and cultivated into DCs with cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4. DCs were harvested after cultivation for 7 days and subjected to irradiation with different dosages of UVB. Then, 200 μg/mL of EGCG was added in certain groups 24 h before irradiation. DCs simply treated with UVB or treated with both UVB and EGCG were co-cultured with lymphocytes, and Mono-nuclear cell direct cytotoxicity (MTT) assay was used to detect the ability of DCs to stimulate proliferation of lymphocytes. Surface markers CD80, CD86, human leukocyte antigen(locus)-DR (HLA-DR), and CD40 were detected using flow cytometry, and the levels of IL-10 and IL-12 secreted from DCs 24 h after cultivation were measured using ELISA.
Results  UVB irradiation was able to inhibit the ability of DCs to stimulate the proliferation of lymphocytes and surface expressions of CD80, CD86, HLA-DR, and CD40 on DCs in a dose-dependent manner. The inhibition rate of DCs was improved to some extent after treatment with 200 μg/mL of EGCG. UVB showed no significant influence on the secretion of IL-10 and IL-12 from DCs, while EGCG was able to down-regulate the secretion level of IL-12 and up-regulate that of IL-10.
Conclusions  EGCG can antagonize the inhibitory effect on DCs induced by UVB irradiation. This function has some relationship with its protecting effect of the expression of the costimulating molecules on the surface of DCs and the secretion level of IL-10 and IL-12.     

Effects of monomethylfumarate on dendritic cell differentiation

BACKGROUND: Fumaric acid esters (FAE) are effective against psoriasis vulgaris and monomethylfumarate (MMF) is believed to be the most bioactive metabolite of this medication. Earlier we found that the beneficial effects of FAE medication are accompanied by a downregulation of type 1 cytokine production by T-helper (Th) lymphocytes, which are important as they maintain a type 1 cytokine [interferon (IFN)-gamma, interleukin (IL)-2] environment in the skin lesions of psoriasis vulgaris patients and once maximal beneficial effects are obtained type 2 cytokine production is also decreased. In vitro MMF selectively induced type 2 cytokine production by primed Th lymphocytes, whereas type 1 cytokine production by and profileration of T lymphocytes were unaffected. OBJECTIVES: As dendritic cells (DCs) present in these skin lesions play a key role in the activation of Th lymphocytes, we investigated the effects of MMF on monocyte-derived DC differentiation. METHODS: Monocytes were differentiated into immature (i) DCs by cytokines with or without MMF. To establish whether these cells were differentiated into iDCs, we analysed the expression of cell surface molecules on these cells and the capacity to capture antigens using flow cytometry. Next, we determined whether these MMF-incubated (MMF-)iDCs could be matured by lipopolysaccharide (LPS) and whether MMF affected this responsiveness as well. For this purpose we measured cytokine production by these LPS-stimulated cells (MMF-DCs) using enzyme-linked immunosorbent assays as well as their ability to activate naive Th lymphocytes. RESULTS: The presence of MMF during the differentiation of monocytes into iDCs resulted in cells that retained low levels of CD14 and hardly expressed CD1a. Upon maturation, these MMF-iDCs upregulated CD83 and costimulatory molecules and HLA-DR on their surface, indicating that these cells respond to LPS, albeit less than control iDCs. In addition, in response to LPS, MMF-iDCs did not decrease the capacity to capture antigens when compared with control iDCs. MMF-DCs hardly produced IL-12p70 and IL-10 and low levels of tumour necrosis factor (TNF)-alpha, whereas IL-8 produced by MMF-DCs and control DCs did not differ. Moreover, MMF-DCs were less able to induce IFN-gamma production by naive Th lymphocytes compared with control DCs. The production of IL-4 and IL-10 by naive Th lymphocytes cocultured with MMF-DCs did not differ from that by T cells cocultured with control DCs. CONCLUSIONS: MMF inhibited the monocyte-derived DC differentiation resulting in cells that cannot be appropriately matured to DCs. Consequently, these MMF-DCs are less effective than control DCs in stimulating type 1 cytokine, but not type 2 cytokine production, in Th lymphocytes. This general immunomodulatory effect may in part explain the beneficial effects of FAE therapy in psoriasis.     

The maturation-dependent production of interleukin-16 is impaired in monocyte-derived dendritic cells from atopic dermatitis patients but is restored by inflammatory cytokines TNF-alpha and IL-1beta

BACKGROUND: Maturation of dendritic cells (DCs) influences important DC functions such as production of cytokines. Recently, DCs were identified as a source of interleukin-16 (IL-16), a chemotactic factor for DCs themselves, CD4+ T cells, and eosinophils. There is evidence that DC-derived IL-16 may contribute to the pathogenesis of atopic dermatitis (AD). OBJECTIVE: To investigate the production of IL-16 during differentiation of monocytes into DCs in healthy individuals and patients with AD. METHODS: IL-16 production was investigated by quantitative real-time RT-PCR, intracellular cytokine staining, immunoblotting, and ELISA. RESULTS: DCs generated from peripheral monocytes by 5-day culture in the presence of IL-4 and granulocyte/macrophage colony-stimulating factor acquired the capability to synthesize, store, and secrete IL-16. Storage and release of IL-16 was further enhanced during final DC maturation induced by additional 3-day culture with tumor necrosis factor-alpha (TNF-alpha) and monocyte-conditioned medium. Maturation, as determined by up-regulation of CD83 and CD86 surface expression, and production of IL-16, but not production of IL-10 and IL-12p40 was impaired in day 8 DCs derived from AD patients compared to those from healthy donors. Stimulation of day 8 DCs from AD patients with TNF-alpha and IL-1beta enhanced the expression of CD83 and CD86 and restored the production of IL-16. CONCLUSIONS: Signals involved in the activation and maturation of DCs enhance their capacity to produce IL-16. Functional abnormalities present in patients with AD at the monocyte level may account for impaired maturation and IL-16 production of monocyte-derived DCs.     

中药汽疗联合阿维A和复方甘草酸苷治疗寻常性银屑病的疗效观察

目的评价中药汽疗联合阿维A和复方甘草酸苷治疗寻常性银屑病的临床疗效。方法将入选的256例银屑病患者随机分为治疗组和对照组。治疗组应用中药汽疗联合口服阿维A胶囊和复方甘草酸苷片,对照组仅口服药物,疗程8周。结果治疗组有效率为84.62%,对照组为69.84%,两组比较差异有统计学意义(P<0.05)。结论中药汽疗联合阿维A和复方甘草酸苷治疗寻常性银屑病疗效好,安全可靠。     

寻常型银屑病患者几种血清细胞因子的检测

目的:探讨血清中白介素12(IL-12)、IL-18、IL-4及IL-10水平与寻常型银屑病的发病关系。方法:采用双抗体夹心ELISA法检测30例寻常型银屑病患者及15例健康志愿者血清IL-12、IL-18、IL-4及IL-10的水平。结果:银屑病组IL-18水平显著增高,IL-10水平显著降低,与健康对照组相比差异均有统计学意义(P<0.05)。结论:IL-18、IL-10在银屑病的发病中可能发挥重要作用。     

促炎细胞因子对寻常性银屑病患者外周血淋巴细胞表达DARC的影响

目的研究促炎性细胞因子对寻常性银屑病患者外周血淋巴细胞表达达菲抗原趋化因子受体(Duffy antigen/receptor for chem okines,DARC)的影响,探讨DARC在银屑病发病机制中的作用。方法分离、纯化寻常性银屑病患者外周血淋巴细胞,用促炎性细胞因子IL-1β、IL-6或TNF-α刺激,应用FACS技术和RT-PCR方法检测寻常性银屑病患者外周血淋巴细胞DARC蛋白和mRNA表达。结果寻常性银屑病患者外周血淋巴细胞在受到促炎性细胞因子IL-1β、IL-6或TNF-α刺激后,细胞表面DARC蛋白的表达明显降低(P<0.01),细胞内DARC mRNA表达增加。其中以进行期寻常性银屑病患者外周血淋巴细胞DARC表达的改变更为明显。结论促炎性细胞因子能调节银屑病患者外周血淋巴细胞表面DARC蛋白和细胞内DARC mRNA的表达。     

不同证候银屑病患者外周血Th1/Th17细胞表达差异的研究

目的 探讨不同证候银屑病的发病与外周血Th1/Th17细胞轴漂移的相关性。方法 用流式细胞仪检测CD4+T细胞内细胞因子干扰素?酌（IFN-?酌）、白介素17A（IL-17A）的表达情况,双抗体夹心法（ELISA）检测不同证候银屑病患者血清中Th１型细胞因子IFN-?酌和Th17型细胞因子IL-17A的水平。 结果 血热组银屑病患者外周血单一核细胞（PBMC）中CD4+IFN-?酌+水平显著高于血瘀组及健康对照组（P < 0.05）,而血瘀组与健康对照组外周血PBMC中CD4+IFN-?酌+水平差异无统计学意义。血热组银屑病患者PBMC中CD4+IFN-?酌+水平与PASI评分呈正相关（P < 0.05）,血瘀组不具相关性（P > 0.05）。血热组、血瘀组银屑病患者及健康对照组PBMC中CD4+IL-17A+三组间差异无统计学意义。血热组银屑病患者PBMC中CD4+IL-17A+水平与PASI评分不具相关性（P > 0.05）,而血瘀组呈正相关（P < 0.05）。ELISA结果示,血热组银屑病患者外周血清中IFN-?酌水平显著高于血瘀组及健康对照组（P < 0.05）,而血瘀组与健康对照组差异无统计学意义（P > 0.05）;血热组银屑病患者血浆中IFN-?酌水平与PASI评分呈正相关（P < 0.05）,血瘀组银屑病患者呈负相关（P < 0.05）。血热组、血瘀组银屑病患者及健康对照血清中IL-17A水平三组间差异无统计学意义（P > 0.05）,血热、血瘀组银屑病患者血清中IL-17A水平与PASI评分不具相关性（P > 0.05）。 结论 Th1细胞在血热型银屑病的发病中占主导地位,当血热型银屑病向血瘀型转化或正常转归时,Th1细胞的表达下降,而Th17细胞在不同证候银屑病患者中差异无统计学意义。     

The antipsoriatic effect of thiamazole is not accompanied either by significant changes in blood lymphocyte subsets nor by serum concentration of TNF-alpha

It has been reported that the antithyroid thiourethylenes, propylthiouracil and methimazole can bring about significant clinical improvement in patients with psoriasis vulgaris. In this paper, we reconfirmed the clinical usefulness of the antithyroid drug thiamazole, which is similar to methimazole, in Japanese patients with psoriasis vulgaris. We further asked whether the clinical improvement was linked to the changes in some immunological parameters or not. To answer this question, we analyzed peripheral blood lymphocyte subsets and measured serum concentrations of TNF-alpha and IL-1beta in 13 patients with psoriasis vulgaris before and during thiamazole administration. The mean percentage of CD4+ HLA-DR+ lymphocytes in the patients was constantly elevated before and 12 weeks after the administration. The patients treated with thiamazole showed a significant increase in the ratio of CD4+/CD8+ at the 4th week, though it declined at the 8th and 12th week. Other lymphocyte subsets showed no significant change during the treatment. The concentrations of serum TNF-alpha exhibited no significant change during thiamazole administration. Serum concentration of IL-1beta was under the detection level. These results indicate that thiamazole may improve the psoriatic lesion via other immune mechanisms, or the effects can not be reflected by the peripheral lymphocyte subsets and cytokine which we investigated.