抗P选择素功能域单抗对人树突状细胞表型及IL-12分泌的影响

观察抗P选择素Lectin EGF功能域单抗 (PsL EGFmAb )对体外培养人树突状细胞 (DC )表型以及促炎细胞因子IL 1 2分泌的影响 ,探讨PsL EGFmAb对DC炎性成熟过程中的调节作用。通过SCF、GM CSF、TGF β1 、Flt 3和TNF α体外培养体系 ,从脐血CD34+ 造血干细胞中诱导扩增获得DC ,并于成熟中用PsL EGFmAb进行干预。采用流式细胞仪分析细胞表型CD1a、CD1 1c、CD83、CD80、CD86和HLA DR ;采用RT PCR检测IL 1 2p35、p4 0mRNA表达 ;以及ELISA法测定IL 1 2p70分泌的含量。结果显示 ,PsL EGFmAb可下调成熟中DC表面CD1 1c、CD83、CD80、CD86和HLA DR的表达 ,同时能抑制DC内IL 1 2p35、p4 0mRNA的转录和IL 1 2p70的分泌。本研究提示 ,PsL EGFmAb对DC黏附共刺激分子表达和促炎细胞因子合成具有抑制作用 ,并可能影响和调抑DC成熟及其提呈抗原功能     

IL-13抑制人肾小球系膜细胞IL-12的表达

为了探讨白细胞介素 13(IL 13)对体外培养的人肾小球系膜细胞产生白细胞介素 12 (IL 12 )的影响。我们用脂多糖(LPS 10 μg/ml) ,不同浓度的IL 13对系膜细胞培养 ,分别采用ELISA法和半定量RT PCR法检测细胞上清液的IL 12和系膜细胞IL 12p4 0mRNA表达。结果提示 5 %NCSRPMI 16 4 0基础培养条件下的系膜细胞未检测到IL 12蛋白分泌及其mRNA表达。在LPS刺激下系膜细胞的IL 12p4 0mRNA表达加强 ,并分泌出大量的IL 12。IL 13在 1～ 10 0ng/ml浓度范围内对LPS诱导的系膜细胞IL 12分泌及IL 12p4 0mRNA表达的抑制作用呈剂量依赖趋势。本研究认为IL 13可能通过抑制IL 12的产生 ,而调整了体内Th1/Th2细胞因子平衡 ,作为抗炎性细胞因子在肾小球肾炎发病机制中发挥一定作用     

LPS持续刺激对小鼠骨髓树突状细胞成熟的影响

目的　研究LPS持续刺激对小鼠骨髓树突状细胞 (DC)成熟的影响。方法　小鼠骨髓细胞用GM CSF培养 7d ,持续刺激组全程加入LPS ,短期刺激组在最后 48h加入LPS ,对照组不加LPS。流式细胞仪检测细胞表型和细胞摄取抗原的能力 ,ELISA检测细胞产生的细胞因子 ,混合淋巴细胞培养检测细胞提呈抗原的能力。结果　用LPS持续刺激的小鼠骨髓DC表达MHCⅡ、CD86、CD80和CD11c等分子和分泌TNF α和IL 12 (p70 )的能力并未增加 ,吞噬FITC OVA的能力显著升高 ,刺激同种异基因T细胞和刺激同种同基因T细胞增殖的能力亦显著低于LPS短期刺激组。结论　LPS持续刺激可抑制DC的发育成熟 ,可能是持续严重感染时免疫功能低下的原因     

人外周血及脐血树突状细胞的体外分离培养

取正常人外周血或脐血,经淋巴细胞分离液分离,取中间白膜层,培养板中进行粘附,粘附细胞加培养液和细胞因子（外周血加GM－CSF和IL－4,脐血加GM－CSF和TNF－α）培养,对其形态、表型和功能分别进行鉴定和测定。结果表明约经过1周左右培养,悬浮细胞表现为典型的DC形态,带有毛刺样凸起,经DC单克隆抗体染色后用流式细胞仪测定脐血72％为DC,外周血93％为DC,并且可以刺激同种异体淋巴细胞的增殖反应。所以通过这样的不同细胞因子组合可以从人外周血和脐血中诱导培养出大量的DC细胞,为其进一步的研究奠定了基础。     

新生儿脐血单核细胞在脂多糖刺激下分泌IL-6及出现IL-6mRNA表达的实验研究

目的 :通过观察LPS对新生儿脐血单个核细胞 (MC)分泌IL 6及表达IL 6mRNA基因的影响 ,探讨严重细菌感染时新生儿机体防御反应机制。方法 :取肝素抗凝剂脐血 ,用密度离心分离法分离MNC ,以RPMI16 40培养液调整细胞浓度为1× 10 6 ml- 1 ,将细胞悬液铺于 2 4孔培养板上 ,依次加入不同浓度脂多糖 (LPS)培养 36h或同一浓度LPS(1μg ml)培养不同时间 ,收集培养上清液及细胞 ,分别用ELISA和RT PCR方法测定IL 6及IL 6mRNA表达情况。结果 :①脐血MNC在LPS刺激 3、6、12、18、2 4、36h后IL 6分泌水平逐步增高 ,6h以后增加尤为明显 ,与其他各时间点比较有非常显著的差异 (P <0 0 0 1)。LPS刺激组与无LPS对照组相同时间点比较 ,6h内IL 6变化水平无差异 ,6h以上各点有显著差异 (P <0 0 1)。RT PCR方法检测显示LPS刺激后 3h即可见IL 6mRNA基因表达。②脐血MNC受不同浓度LPS刺激时 ,IL 6分泌水平随LPS浓度递增。③全部脐血MNC均检测到IL 6mRNA基因表达。结论 :LPS能诱导新生儿脐血MNCIL 6mRNA基因转录 ,从而促使IL 6合成、分泌 ,该作用呈时间、剂量依赖性变化。     

用含有特定细胞因子的无血清培养基培养慢性乙型肝炎病人的树突状细胞

目的　体外扩增慢性乙肝病人的树突状细胞 (dendriticcell,DC) ,从细胞表型和功能上鉴定和研究。方法　用含GM CSF和IL 4的无血清培养基AIM V体外培养慢性乙肝病人外周血单个核细胞 ,获得树突状细胞。流式细胞仪检测细胞表型 ,IL 1 2ELISA试剂盒检测DC分泌IL 1 2的水平 ,并观察加入细胞因子TNF α后对DC培养的影响。结果　慢性乙肝病人的外周血单个核细胞用AIM V培养及细胞因子诱导后 ,经贴壁法纯化 1 0 0mL可获得 0 .5× 1 0 7～ 1 .5× 1 0 7成熟的具有典型形态的DC ,加入TNF α后 ,CD83阳性占 60 .80 % ,明显高于未加组 (P <0 .0 5) ,IL 1 2分泌较未加TNF α组增高近 1 0倍。结论　①慢性乙肝病人的DC可用AIM V无血清培养基及特定的细胞因子诱导在体外大量获得 ,TNF α是诱导DC成熟的重要的细胞因子。②典型的细胞形态和CD1 4 -、HLA DRhigh+、CD86high+的细胞表面分子特征可作为临床上快速鉴定培养DC的标志。     

CpG寡聚脱氧核苷酸(CpG ODN)协同促进脂多糖诱导的小鼠巨噬细胞增殖与迁移

目的探讨CpG寡聚脱氧核苷酸(CpG ODN)对脂多糖(LPS)诱导的巨噬细胞增殖与迁移能力的影响及机制。方法使用1 mg/L LPS处理小鼠RAW264.7巨噬细胞建立体外炎症细胞模型,采用CCK-8法检测CpG ODN(500 nmol/L)对LPS诱导的巨噬细胞增殖的影响,采用Transwell~(TM)实验检测CpG ODN对细胞迁移能力的影响;采用Western blot法检测p38丝裂原激活蛋白激酶(MAPK)、 c-Jun氨基末端激酶(JNK)、胞外信号调节激酶(ERK)、核因子κBp65(NF-κB p65)的蛋白磷酸化水平,同时使用以上通路的抑制剂SB203580、 SP600125、 PD98059、 BAY11-7082,探讨CpG ODN发挥效应的机制;采用实时定量PCR检测CpG ODN对LPS诱导产生的单核细胞趋化蛋白1(MCP-1)、环加氧酶2(COX2)mRNA水平的影响。结果 CpG ODN协同促进LPS诱导的巨噬细胞增殖与迁移,并促进COX2、 MCP-1的转录,增强JNK、 ERK信号通路蛋白的磷酸化水平,并且JNK与ERK信号通路激酶抑制剂可有效降低CpG ODN的协同效应。结论 CpG ODN可通过JNK与ERK途径协同促进LPS诱导的巨噬细胞增殖与迁移并促进COX2、 MCP-1的转录。     

IL-37抑制脂多糖诱导的小鼠树突状细胞活化

目的探讨白细胞介素37(IL-37)对细菌脂多糖(LPS)诱导的小鼠树突状细胞(DC)活化的调节作用。方法应用GM-CSF和IL-4诱导小鼠骨髓细胞向DC分化,抗CD11c磁珠分选DC。IL-37预处理DC后,进行LPS刺激。流式细胞术检测DC表面共刺激分子(CD80、CD86)表达水平,实时荧光定量PCR检测肿瘤坏死因子α(TNF-α)、IL-6和IL-1αmRNA表达水平,流式细胞微球芯片试剂盒(CBA试剂盒)检测细胞培养上清中IL-1α、IL-6、TNF-α等因子的浓度。结果 DC诱导成功,磁珠分选能够获得高纯度的DC(>90%)。IL-37降低LPS诱导的DC表面共刺激分子CD80、CD86的表达,并抑制DC合成IL-1α、IL-6、TNF-α。结论 IL-37可以通过降低共刺激分子和炎症因子的表达抑制LPS刺激的DC活化。     

IL-17A 协同GM-CSF 和LPS 促进骨髓细胞衍生树突状细胞的分化和成熟研究

目的:探讨IL鄄17A 对小鼠骨髓细胞衍生树突状细胞分化和成熟的影响。方法:分离小鼠骨髓细胞,加入含GM-CSF(20 ng/ ml)RPMI1640 完全培基培养8 d,诱导小鼠骨髓单个核细胞向DC 分化,加入LPS(1 滋g/ ml)继续培养36 h,进一步诱导DC 成熟,同时在骨髓细胞衍生诱导DC 分化及成熟的不同阶段加入不同浓度的rmIL-17A(10、100 ng/ ml),采用流式细胞术检测DC 表面共刺激分子的表达,ELISA 方法检测DC 培养上清中IL-12p40 和IL-10 水平。结果:rmIL-17A 可促进GM-CSF 诱导骨髓细胞衍生DC 表面共刺激分子CD40、CD80、CD86 和MHC域的表达,且具有剂量依赖性,其中以高浓度rmIL-17A刺激组的CD40 及MHC域表达增加最显著;在LPS 诱导DC 成熟阶段加入rmIL-17A,骨髓细胞衍生DC 共刺激分子CD40、CD80、CD86 和MHC域的表达均明显增加,并且随着rmIL-17A 浓度的增加,CD86 和MHC域的表达水平也随之增高;同时与未加rmIL鄄17A 的对照组相比,低浓度rmIL-17A 组LPS 刺激骨髓细胞衍生DC 分泌IL-12p40 和IL鄄10 水平均显著增加(P <0.001),高浓度rmIL-17A 组IL-12p40 水平显著增高(P<0.001),但IL-10 水平没有变化。结论:IL-17A 可促进GM-CSF 诱导的骨髓细胞衍生DC 前体细胞表型发展,并能协同LPS 诱导骨髓衍生DC 的分化和成熟。     

诱导实体瘤细胞生成具有树突状细胞样的抗原提呈细胞

目的　探讨体外用细胞因子诱导实体瘤细胞生成具有树突状细胞样的抗原提呈功能。方法　以人肝母细胞瘤为研究模型 ,用人胰岛素样生长因子 (IGF 1)、人重组粒细胞单核细胞集落刺激因子 (GM CSF)、人重组白细胞介素 4 (IL 4 )及肿瘤坏死因子α(TNF α)诱导培养瘤细胞 2周 ,用流式细胞仪及MTT法检测诱导前后瘤细胞表型及抗原提呈功能。结果　用细胞因子处理瘤细胞后 ,树突状细胞样的特异性标志CD1a和CD83表达阳性率显著上调 ;MHCⅡ、B7 1、B7 2、ICAM 1和CD4 0表达水平显著增加。同时 ,瘤细胞能显著刺激同种异体淋巴细胞增殖 ,促进IL 12的产生 ,明显提高细胞毒性T淋巴细胞 (CTL)对瘤细胞的杀伤活性。结论　初步实验结果提示 ,体外用IGF 1、GM CSF、IL 4及TNF α等细胞因子能诱导肝母细胞瘤细胞生成具有树突状细胞样特征的抗原提呈细胞。     

人IL-12的克隆、表达及生物活性的鉴定

目的：克隆中国人ＩＬ－２ｐ４０基因,构建ＩＬ－１２的真核基因表达载体。方法：采用ＲＴ－ＰＣＲ从北京地区人脐血树突状细胞（ＤＣ）中克隆ＩＬ－１２ｐ４０ｃＤＮＡ基因,并进行序列分析。利用ｐｃＤＮＡ３．１和ｐＬＸＰＸＳＮ构建ＩＬ－１２表达载体,转染人肝癌细胞后对其进行生物学和免疫学分析。结果：北京地区人ＤＣ中ＩＬ－１２ｐ４０ｃＤＮＡ基因２１０位密码子有独特的结构,为ＧＣＣ（Ａｌａ）。并且２３９和２９１位     

Passive transfer of flt-3L-derived dendritic cells delays diabetes development in NOD mice and associates with early production of interleukin (IL)-4 and IL-10 in the spleen of recipient mice

CD11c+/CD11b+dendritic cells (DC) with high levels of major histocompatibility complex (MHC) class II and co-stimulatory molecules have been derived from spleen cells cultured with granulocyte-macrophage colony stimulating factor (GM-CSF) + flt-3L + interleukin (IL)-6 (flt-3L-DC). Investigating in vivo the function of DC in non-obese diabetic mice (NOD), we showed that a single injection of this in vitro-derived subset of DC prevents the development of diabetes into prediabetic female mice. In contrast, DC derived from bone marrow cells cultured with GM-CSF + IL-4 [bone marrow (BM)-DC] induced no protection. Moreover, protection against diabetes following injection of flt-3L-DC was associated with IL-4 and IL-10 production in the spleen and the pancreatic lymph nodes of recipient mice, indicating that this DC population is able to polarize the immune response towards a Th2 pathway. As we shown previously, NOD BM-DC exhibit an enhanced capacity to produce IL-12p70 in response to lipopolysaccharide (LPS) and anti-CD40 stimulation compared to BM-DC from control mice. In contrast, NOD flt-3L-DC, as their control mouse counterpart, produced no IL-12p70 to these stimuli. Our findings show that a subset of DC, characterized by a mature phenotype and the absence of IL-12p70 production can be derived from NOD mouse spleen favouring IL-4 and IL-10 regulatory responses and protection from diabetes development.     

转染MHCII类基因对卵巢癌细胞株免疫特性的影响

建立稳定表达CIITA基因的人卵巢癌细胞株HO CIITA ,分析转染CIITA基因对T细胞体外抗肿瘤免疫应答的影响。以CIITA基因逆转录病毒 (pLXSN/CIITA )转染并筛选获得HO CIITA。用FACS和RT PCR分析HLA以及抗原加工递呈基因表达水平。以磁珠法分离得到正常人外周血CD4 +/CD8+T细胞 ,分别进行混合淋巴细胞反应及细胞因子测定 (ELISA和RT PCR )。结果显示 ,转染CIITA基因后 ,使HO细胞HLAII类分子和LMP7基因表达增高 ,而Ii基因表达由阳性转为阴性 ,未检测到TAP1表达 ;HO和HO CIITA细胞刺激CD4 +T细胞分泌IL 4含量两者有显著差异。刺激 4 8h后达到顶峰 ,前者分泌量约为后者的 1/2 ,但分泌IFN γ无差异 ,RT PCR与ELISA两种测定结果一致。表明转染CIITA基因可增加肿瘤细胞表面MHCII类分子的表达 ,该作用与IFN γ具有协同效应 ;并能诱导CD4 +T细胞表达IL 4。     

Stress‐activated dendritic cells interact with CD4+ T cells to elicit homeostatic memory

Evidence is presented that thermal or oxidizing stress‐activated DC interact with CD4⁺ T cells to induce and maintain a TCR‐independent homeostatic memory circuit. Stress‐activated DC expressed endogenous intra‐cellular and cell surface HSP70. The NF‐κB signalling pathway was activated and led to the expression of membrane‐associated IL‐15 molecules. These interacted with the IL‐15 receptor complex on CD4⁺ T cells, thus activating the Jak3 and STAT5 phosphorylation signalling pathway to induce CD40 ligand expression, T‐cell proliferation and IFN‐γ production. CD40 ligand on CD4⁺ T cells in turn re‐activated CD40 molecules on DC, inducing DC maturation and IL‐15 expression thereby maintaining the feedback circuit. The proliferating CD4⁺ T cells were characterized as CD45RA^? CD62L⁺ central memory cells, which underwent homeostatic proliferation. The circuit is independent of antigen and MHC‐class‐II‐TCR interaction as demonstrated by resistance to TCR inhibition by ZAP70 inhibitor or MHC‐class II antibodies. These findings suggest that stress can activate a DC‐CD4⁺ T‐cell interacting circuit, which may be responsible for maintaining a homeostatic antigen‐independent memory.     

逆转录病毒介导乙型肝炎病毒C基因在树突状细胞中的转移

目的：探讨逆转录病毒介导乙型肝炎病毒核心抗原(HBcAg)基因在骨髓来源的树突状细胞(DC)的转移效率，以及对DC成熟和功能的影响。方法：用含有HBV C基因的重组逆转录病毒载体，感染处于分裂期的小鼠骨髓祖细胞，感染后的骨髓细胞在GM—CSF和IL—4存在的条件下继续培养获得成熟的DC，用PCR、RT—PCR，分析目的基因的整合与转录；用Western blot和流式细胞仪，分析HBcAg的表达及基因转移效率，以及检测感染前后DC CD80和MHC—Ⅱ类分子的表达以及分泌IL—12能力的变化；通过将基因转移后的DC与淋巴细胞进行混合培养，检测其体外诱导CTL的能力。结果：逆转录病毒感染后并不影响骨髓来源的DC成熟，对其表面分子CD80和MHC-Ⅱ类分子的表达和分泌IL-12的能力没有影响。目的基因能整合到DC的基因组DNA中，并能转录和翻译。约28％的DC能表达HBcAg，感染后的DC体外能诱导T细胞应答。结论：逆转录病毒能有效地将HBV C基因转移到骨髓来源的DC中，对DC的成熟和功能无明显影响，并能诱导CTL应答，这将有助于开展以DC为基础的慢性乙肝的免疫治疗。     

Small interference RNA modulation of IL-10 in human monocyte-derived dendritic cells enhances the Th1 response

RNA interference technology has been used to modulate dendritic cell (DC) function by targeting the expression of genes such as IL-12 and NF-kappa B. In this paper, we demonstrate that transfection of DC with IL-10-specific double strands of small interference RNA (siRNA) resulted in potent suppression of IL-10 gene expression without inducing DC apoptosis or blocking DC maturation. Inhibition of IL-10 by siRNA was accompanied by increased CD40 expression and IL-12 production after maturation, which endowed DC with the ability to significantly enhance allogeneic T cell proliferation. IL-10 siRNA transfection did not affect MHC class II, CD86, CD83, or CD54 expression in mature DC. To further test the ability of IL-10 siRNA-treated DC to induce a T cell response, naive CD4 T cells were stimulated by autologous DC pulsed with KLH. The results indicated that IL-10 siRNA-transfected DC enhanced Th1 responses by increasing IFN-gamma and decreasing IL-4 production. These findings suggest the potential for a novel immunotherapeutic strategy of using IL-10 siRNA-transfected antigen-presenting cells as vaccine delivery agents to boost the Th1 response against pathogens and tumors that are controlled by Th1 immunity.     

IL‐33 promotes DC development in BM culture by triggering GM‐CSF production

Short‐term DC cultures generated with GM‐CSF and other cytokines have markedly improved our ability to study the immunobiology of DC. Here, we tested 65 cytokines individually for their potential to promote the generation of CD11c⁺ cells in a murine BM culture system. In addition to several cytokines known to promote DC survival and/or growth, IL‐33 was found to augment DC development time‐ and dose‐dependently. Although the resulting CD11c⁺ cells generated in the presence of IL‐33 exhibited a typical dendritic morphology, they expressed MHC class II molecules only at modest levels, showed negligible responses to TLR ligands, produced no detectable IL‐12 p70, displayed PD‐L1 and PD‐L2 on the surface, and failed to activate immunologically naïve T cells efficiently. IL‐33‐induced expansion of CD11c⁺ cells was completely blocked by anti‐GM‐CSF mAb, and GM‐CSF mRNA and protein expression in BM culture was markedly elevated by added IL‐33, indicating that IL‐33 promotes in vitro DC generation indirectly by a GM‐CSF‐dependent manner. With regard to the cellular source, IL‐33‐dependent GM‐CSF production was observed exclusively within the CD45⁺/FcεRI⁺ BM population. Not only do our results reinforce the notion that GM‐CSF serves as a primary DC growth factor, but they also reveal a previously unrecognized mechanism supporting DC development.     

CpG ODN促进树突状细胞成熟的实验研究

目的：研究CpG ODN对小鼠骨髓来源的树突状细胞(bone marrow—derived dendritic cells,BMDC)分化成熟的影响。方法：以含非甲基化CpG基序的寡核苷酸(unmethylated CpG motif containing oligonucleotides,CpG ODN)刺激BMDC，流式细胞术检测DC膜表面CD80表达，ELISA检测DC分泌IL-l2水平，MTT法测定CpG ODN活化的DC刺激T细胞培殖能力。结果：CpG ODN可显著刺激DC表达CD80，分泌IL-l2，增强DC刺激T细胞增殖的能力。结论：CpG ODN可以有效促进DC的功能成熟。     

Polarization of naive T cells into Th1 or Th2 by distinct cytokine-driven murine dendritic cell populations: implications for immunotherapy

Dendritic cells (DCs) activate T cells and regulate their differentiation into T helper cell type 1 (Th1) and/or Th2 cells. To identify DCs with differing abilities to direct Th1/Th2 cell differentiation, we cultured mouse bone marrow progenitors in granulocyte macrophage-colony stimulating factor (GM), GM + interleukin (IL)-4, or GM + IL-15 and generated three distinct DC populations. The GM + IL-4 DCs expressed high levels of CD80/CD86 and major histocompatibility complex (MHC) class II and produced low levels of IL-12p70. GM and GM + IL-15 DCs expressed low levels of CD80/CD86 and MHC class II. The GM + IL-15 DCs produced high levels of IL-12p70 and interferon (IFN)-gamma, whereas GM DCs produced only high levels of IL-12p70. Naive T cells stimulated with GM + IL-4 DCs secreted high levels of IL-4 and IL-5 in addition to IFN-gamma. In contrast, the GM + IL-15 DCs induced higher IFN-gamma production by T cells with little or no Th2 cytokines. GM DCs did not induce T cell polarization, despite producing large amounts of IL-12p70 following activation. A similar pattern of T cell activation was observed after in vivo administration of DCs. These data suggest that IL-12p70 production alone, although necessary for Th1 differentiation, is not sufficient to induce Th1 responses. These studies have implications for the use of DC-based vaccines in immunotherapy of cancer and other clinical conditions.