胰岛素样生长因子1诱导脐血CD14^＋单核细胞转化树突状细胞分化成熟的研究

目的 通过研究胰岛素样生长因子1（IGF-1）对脐血CD14^ 单核细胞转化的树突状细胞分化成熟的影响,以探讨IGF-1对新生儿天然免疫的作用。方法 利用无血清,无激素培养系统,体外培养10例健康足月新生儿脐血及10例健康成人外周血CD14^ 单核细胞,观察IGF-1对新生儿脐血CD14^ 单核细胞转化的树突状细胞（monocyte-derived dendritic cell,mDC)表型和功能的影响。结果 与正常成人比较,IL-4 GM-CSF诱导的新生儿脐血mDC中：CD1a^ 细胞百分率和甘露糖受体（MR）阳性细胞百分率明显降低,细胞表面主要组织相容性Ⅱ型抗原（MHCⅡ）和CD40表达明显降低,其吞噬功能,抗原提呈功能明显下降,IGF-1能显著升高脐血mDCMHCⅡ表达,下调甘露糖受体及细胞的吞噬功能,结论 IGF-1体外可促进脐血CD14^ 单核细胞转化的树突状细胞成熟。     

胰岛素样生长因子1诱导脐血CD14+单核细胞转化树突状细胞分化成熟的研究

目的　通过研究胰岛素样生长因子 1(IGF 1)对脐血CD14 单核细胞转化的树突状细胞分化成熟的影响 ,以探讨IGF 1对新生儿天然免疫的作用。方法　利用无血清 ,无激素培养系统 ,体外培养 10例健康足月新生儿脐血及 10例健康成人外周血CD14 单核细胞 ,观察IGF 1对新生儿脐血CD14 单核细胞转化的树突状细胞 (monocyte deriveddendriticcell,mDC)表型和功能的影响。结果　与正常成人比较 ,IL 4 GM CSF诱导的新生儿脐血mDC中 :CD1a 细胞百分率和甘露糖受体 (MR)阳性细胞百分率明显降低 ,细胞表面主要组织相容性II型抗原 (MHCII)和CD4 0表达明显降低 ,其吞噬功能 ,抗原提呈功能明显下降。IGF 1能显著升高脐血mDCMHCII表达 ,下调甘露糖受体及细胞的吞噬功能。结论　IGF 1体外可促进脐血CD14 单核细胞转化的树突状细胞成熟     

Insulin-like growth factor I promotes maturation and inhibits apoptosis of immature cord blood monocyte-derived dendritic cells through MEK and PI 3-kinase pathways

IGF-I has profound effects on the immune system. We previously reported that IGF-I promoted cord blood (CB)-na?ve T-cell maturation and now show that IGF-I promoted maturation of CB monocyte-derived dendritic cells (DC) with up-regulation of CD83, CD86, CD40, and major histocompatibility complex (MHC) class II molecules, and down-regulation of mannose receptor. Furthermore, IGF-I inhibited apoptosis of CB DC and increased the production of tumor necrosis factor alpha (TNF-alpha). These effects were blocked by specific mitogen-activated protein kinase kinase (MEK) inhibitor (PD98059) and phosphoinositol 3-kinase inhibitor (LY294002). PD98059 partially inhibited the IGF-I-induced up-regulation of MHC class II. In contrast, LY294002 was additive in the IGF-I-induced up-regulation of MHC class II. Moreover, LY294002 significantly increased the percentage of late apoptotic cells in CB. These results imply the involvement of different pathways for the differential regulation of co-stimulatory molecule expression and apoptosis. The addition of anti-TNF-alpha did not neutralize the effects of IGF-I on CB DC maturation and apoptosis. On the contrary, neutralizing TNF-alpha significantly increased the IGF-I-induced up-regulation of CD83 and CD40. We conclude that IGF-I has maturation and survival effects on CB DC. These effects are mediated through both MEK and PI 3-kinase pathways but not through the IGF-I induction of TNF-alpha production by the DC.     

维生素A对脐血树突状细胞分化与成熟及其功能的影响

目的　研究维生素A在体内的代谢活性产物视黄酸 (retinoicacid ,RA)对树突状细胞(dendriticcells ,DC)分化、成熟及功能的影响 ,进一步探索维生素A对免疫功能调节的机理。方法　取健康足月儿脐血 9份分离单个核细胞后 ,进行体外培养诱导DC的同时加入生理浓度的RA ,采用流式细胞仪检测DC的表面分子CD1a、CD83、HLR DR以判断RA对DC分化、成熟的影响 ;混合淋巴反应的强度观测RA对DC抗原递呈功能的作用 ;荧光定量PCR法检测细胞因子IL 12p35、IL 12p4 0、IFN γ(Th1)、IL 4、IL 10 (Th2 )mRNA转录水平 ,分析RA对DC诱导Th细胞分化的影响。结果　培养第 6天RA使DC数量显著下降 (P <0 0 0 1) ,培养第 9天 ,DC总数相仿 ,但RA处理后不成熟DC百分比增高 ,而成熟DC百分比降低 ,差异有显著意义 (P <0 0 0 1) ;DC混合淋巴反应的cpm值降低2 9 4 % (P <0 0 0 1)。细胞因子IL 12、IFN γmRNA下降 ,而IL 4、IL 10的mRNA水平升高 ,差异均具有显著意义。结论　维生素A延迟体外培养的脐血单个核细胞向DC的分化和成熟 ,且降低其混合淋巴细胞反应的能力 ,减少Th1细胞因子的产生而增加Th2细胞因子的产生 ,使免疫反应向Th2方向偏移。     

诱导脐血单个核细胞分化为树突状细胞的实验研究

目的：诱导脐血源树突状细胞对恶性血液病脐血移植后的过继免疫治疗十分重要。诱导条件对树突状细胞产率影响很大。本研究观察粒细胞-巨噬细胞集落刺激因子(GM-CSF)、IL-4及TNF-α分阶段诱导脐血单个核细胞来源树突状细胞的效果。方法：通过Ficoll-Hypaque法分离脐血单个核细胞,以GM-CSF、IL-4及TNF-α分阶段诱导脐血来源树突状细胞的生成,培养12 d收获细胞,以光镜和电镜观察其细胞形态学特点,以流式细胞术检测其表面标记。结果：经培养12 d,每2×10 6脐血单个核细胞收获树突状细胞数为(0.46±0.13)×106,CD80+、CD86+细胞比例分别为89.98%±4.32%、86.86%±7.17%,而CD1a+细胞比例43.16%±5.80%,经光镜和电镜下观察细胞具有典型的树突状细胞形态学特征。结论：通过GM-CSF、IL-4及TNF-α的树突状细胞培养体系和分阶段诱导法可高效率诱导脐血树突状细胞。［中国当代儿科杂志,2004, 6(5): 377-380］     

人脐血CD_(34)~ 细胞体外诱导树突状细胞及其功能的检测

目的　探讨人脐血CD 34 细胞体外诱导树突状细胞 (dendriticcells ,DCs)的可能性 ,并检测DCs的功能。方法　利用吸附单克隆抗体 磁珠分离系统 (MACS)获得脐血CD 34 细胞 ,体外以重组的人干细胞集落刺激因子 (hSCF)、人粒 巨噬细胞集落刺激因子 (hGM CSF)、人肿瘤坏死因子α(hTNF α)、人酪氨酸激酶受体家族III的配体 (hFL)诱生DCs。流式细胞仪检测DCs的表型 ,3H TdR掺入法检测DCs体外刺激同种异体T细胞增殖的功能 ,最后通过乳酸脱氢酶法检测DCs活化的T细胞对肿瘤细胞的杀伤性。结果　从人脐血分离到的CD 34 细胞 ,经hSCF、hGM CSF、hTNF α、hFL共同培养后 ,第 7天镜下即可见典型形态的DCs ,第 10天时明显增多 ,细胞表型检测见CD 1a 细胞的比例增加到 (2 8± 4) %(P <0 0 1) ,人类白细胞抗原DR(HLA DR)表达占 (94± 11) %。在三种不同的混和比例下 ,DCs均可刺激异源T细胞增殖 ,其中以DCs∶T细胞为 1∶5时增殖最明显。而且被激活的T细胞对三种不同组织来源的肿瘤细胞均产生了明显的杀伤性 (P <0 0 1)。结论　人脐血CD 34 细胞体外经细胞因子诱导培养 ,可生成大量具有典型功能的成熟DCs。     

Normal monocyte-derived dendritic cell function in patients with Langerhans-cell-histiocytosis

BACKGROUND: Langerhans cell histiocytosis (LCH) is a histiocytic disease, characterized by the lesional accumulation of dendritic Langerhans cells together with T cells and eosinophils. The cause of this disease is unknown. Langerhans cells are bone marrow-derived dendritic cells (DCs), which can develop from CD34(+) hematopoietic progenitor cells as well as from monocytes. PROCEDURE: To test whether LCH patients have a general functional defect present in cells of their DC lineage, we generated immature DCs by culturing monocytes from nine patients with single- or multisystem LCH with GM-CSF and IL-4, and analyzed their phenotype and function before and after an in vitro maturation stimulus. Immature DCs were analyzed for their phenotype and cytokine production, DCs matured in response to TNF-alpha plus PGE(2) were analyzed for their phenotype, their stimulatory capacity in MLR, cell aggregation, and activation-induced apoptosis. RESULTS: In summary, no difference was found between both immature as well as mature DCs generated from patients and controls regarding the expression of CD1a, CD80, CD86, MHC class I, and MCH class II antigens. Similarly, no difference was found regarding IL-10, -12, and TNF-alpha production, as well as regarding cell aggregation and apoptosis in response to external stimuli. CONCLUSIONS: The absence of gross functional abnormalities in DCs generated from monocytes from patients with LCH makes the existence of a severe functional defect affecting all cells of the DC lineage in these patients unlikely.     

Method of birth alters interferon-gamma and interleukin-12 production by cord blood mononuclear cells

Many uncertainties exist regarding the capability of cord blood mononuclear cells (CBMC) to produce cytokines. A number of conflicting reports led us to examine the effects of method of birth on CBMC production of interferon-gamma (IFN-γ), interleukin-4 (IL-4) and interleukin-12 (IL-12). While constitutive production of IL-4 was found in both vaginally and cesarean-delivered infants, constitutive IFN-γ or IL-12 production was found in neither. CBMC from vaginally delivered infants responded to stimulation with concanavalin A/phorbol 12-myristate 13-acetate (Con A/PMA), phytohemagglutinin (PHA), and lipopolysaccharide (LPS) with significantly higher levels of IFN-γ than CBMC from unlabored cesarean section (CS) infants. Production of IL-12 was increased in the vaginally delivered group in response to LPS and PHA but not to ConA/PMA. In contrast, mode of delivery was not associated with differences in IL-4 production. These results indicate that mode of delivery significantly alters the capability of CBMC to produce some cytokines and therefore should be taken into account in interpreting fetal/neonatal mononuclear cell function studies.     

比较卡介苗及其成份对脐血单核细胞转化的树突状细胞成熟的影响

通过比较卡介苗（BCG），卡介苗多糖核酸提取物（BCG-PSN）以及热休克蛋白65（HSP65）对脐血单核细胞转化的树突状细胞成熟的影响。以探讨BCG及其有效成分对新生儿免疫功能的作用。经体外分离，诱导19例健康足月新生儿CD14^ 单核细胞分化成为未成熟树突状细胞，并观察BCG，BCG-PSN和HSP65对其成熟的影响。结果：BCG可通过上调CD83，CD86和MHCⅡ类抗原，下调甘露聚糖受体（MR）促进脐血单核细胞转化的树突状细胞成熟，BCG-PSN和HSP65不能促进脐血树突状细胞分化成熟，提示BCG可能通过促进脐血树突状细胞的成熟，影响新生儿T细胞功能发育，而BCG-PSN和HSP65并无此作用。     

人脐血CD34^＋细胞体外诱导树突状细胞及其功能的检测

目的探讨人脐血CD     

不同方式刺激脐血树突状细胞对细胞因子诱导杀伤细胞和自然杀伤细胞杀伤活性的影响

目前对造血干细胞移植(hematopoietic stem cell transplantation,HSCT)后残留肿瘤细胞的清除需通过过继免疫治疗等手段来解决.通过特定细胞因子联合扩增脐血中细胞因子诱导杀伤(cytokine-induced killer,CIK) 细胞、自然杀伤(natural killer,NK)细胞,可增强脐血移植(UCBT)后的移植物抗白血病(graft-versus-leukemia,GVL)效应.树突状细胞(dendritic cells ,DC)在免疫应答的诱导中具有独特地位,     

Diminished response to interleukin-10 and reduced antibody-dependent cellular cytotoxicity of cord blood monocyte-derived macrophages

Monocyte-derived macrophage (MPhi) subsets are generated by antagonistic induction pathways. A helper MPhi-type (Mh-MPhi) is induced by interferon gamma (IFN-gamma), whereas a cytotoxic MPhi-type (Mc-MPhi), induced by interleukin-10 (IL-10), is a potent mediator of antibody-dependent cellular cytotoxicity (ADCC). Compared with MPhi from healthy adults [peripheral blood monocyte-derived macrophages (PBMPhi)], cord blood MPhi (CBMPhi) were found less capable of generating Mh-MPhi. Here we tested the hypothesis that their generation of Mc-MPhi via IL-10 is also impaired. MPhi surface markers were phenotyped. IL-10 protein and mRNA production were detected after stimulation [alphaCD3 monoclonal antibody (mAb)]. CBMPhi or PBMPhi were co-cultured with MPhi-depleted mononuclear cells of adults and CD4-targeting antibodies as models for ADCC were added. In cord blood, we found diminished alphaCD3-induced IL-10 protein and mRNA production (p < 0.05 versus adults). Basal CD16 and HLA-DR expressions on CBMPhi of preterm and full-term neonates were lower (p < 0.05 versus PBMPhi). IL-10 had reduced effects on CD16 up- and HLA-DR down-modulation on CBMPhi (p < 0.05 versus PBMPhi). CD4-directed receptor modulation and deletion were reduced in the presence of CBMPhi (p < 0.05 versus PBMPhi). IL-10 failed to enhance their ADCC capacity, which was in contrast to PBMPhi (p < 0.05). These data suggest that CBMPhi have an impaired cytotoxic capacity via lower sensitivity toward IL-10.     

人脐血间充质干细胞对脐血CD+34细胞体外扩增作用的研究

目的 探讨含人脐血来源的间充质干细胞(MSCs)体系在体外对脐血造血干细胞(HSCs)扩增作用。方法 (1)用含人脐血MSCs及不同造血生长因子(HGFs)组合的无血清扩增体系对人脐血CD34^ 细胞进行体外扩增。(2)于扩增前及扩增后第6、12天分别用双色流式细胞仪动态检测HSCs表面抗原标记：CD34^ 、CD34^ CD38^-、CD34^ CD3^ 、CD34^ CD19^ 、CD34^ 和CD34^ CD41^ 。细胞的含量。(3)按本实验室方法行体外半固体培养,观察扩增前后脐血细胞粒-单核细胞集落形成单位(CFU-GM)、爆式红系集落形成单位(BFU-E)、混合集落形成单位(CFU-Mix)及高增殖集落形成单位(CFU-HPP)集落形成情况。结果(1)含人脐血MSCs体系对脐血CD34^ CD38^ 细胞的扩增倍数在第6天和第12大分别为159和437倍。该业群百分比在单纯因子组扩增第12天时为1．98％,而在含脐血MSCs组为9．98%,明显高于扩增前。(2)集落培养表明,含脐血MSCs组扩增第12天与扩增第6天相比,其CFU-Mix和CFU-HPP的扩增倍数增加,而单纯因子组这两种集落的扩增倍数下降。(3)随扩增天数的增加,两组扩增体系中CD34^ CD3^ 和CD34^ CD41a^ 细胞均明显增加,而CD34^ CD19^ 和CD34^ CD3^ 细胞均明显减少。两组相比,含脐血MSCs组差异更显著。结论 (1)含脐血MSCs体系不仅能扩增更原始的造血干／祖细胞(HSPC),且具有在短期内(12d)保持HSCs不耗竭。(2)含脐血MSCs体系对脐血CD34^ 细胞向定向祖细胞的扩增,主要为髓系及巨核系祖细胞,而对其向淋巴系祖细胞的扩增具有抑制作用。     

脐血巨核细胞体外扩增及其功能的研究

目的研究扩增后脐血巨核细胞的生物学特性和功能,为脐血巨核细胞的扩增和临床应用提供依据。方法收集足月妊娠健康新生儿的脐带血,免疫磁珠法分离出其中的CD34^＋细胞。采用血小板生长因子（thrombopoietin,TPO）＋干细胞因子（stem cell factor,SCF）＋白细胞介素3（interleukin-3,IL-3）＋IL-6和TPO＋SCF两种细胞因子组合,将富集的脐血CD34^＋接种于无血清无基质细胞的悬浮培养体系中,分别在3、7、10、14d收集扩增产物。运用流式细胞术检测巨核细胞的表型;血浆块法检测巨核细胞集落（colony forming unit-megakaryocyte,CFU-MK）的形成;对巨核细胞进行DNA含量检测以评价其成熟程度;血小板体外活化实验及SCID小鼠体内移植实验评价扩增后巨核细胞的功能。结果不同细胞因子组合和培养时间扩增后,巨核细胞的数量和生物学特性不同。随着培养时间的延长,巨核细胞（CD41^＋）的数量逐渐增加,但培养至14d时增势减缓。因子组合TPO＋SCF＋IL-3＋IL-6各时间段的扩增能力（分别扩增5．2、40．7、121．2、149．7倍）均比因子组合TPO＋SCF（分别扩增3．8、27．4、85．9、106．5倍）强,但因子组合TPO＋SCF的扩增能力仍能满足临床的需要。巨核祖细胞（CD34^＋CD41^＋）的数量在第7天时最多（分别增加43．4和36．2倍）,这也被CFU-Mk所证实。DNA含量检测发现,随着培养天数的增加,多倍体细胞所占的百分比增加。体外血小板活化实验证实,扩增的巨核细胞在体外可产生血小板,有正常巨核细胞功能。移植后两组小鼠的骨髓中均检测到人CD45^＋和CD41^＋细胞。小鼠外周血中人血小板在移植后3d就可测到,5d就可达到高水平（分别为20．7％和17．9％）,维持20d以后才逐渐下降。结论通过对扩增后巨核细胞的生物学特性的研究,有助于寻找有效、简便、易于植入受者体内的扩增方法。体外扩增的脐血巨核细胞可植入骨髓并产生功能正常的血小板。     

Cytotoxic function of umbilical cord blood natural killer cells: relevance to adoptive immunotherapy

Decreased graft-versus-host disease (GVHD), ease of accessibility, and sustained engraftment encourage the use of umbilical cord blood (UCB) as an alternative source to bone marrow for immune reconstitution in children with leukemia. Natural killer (NK) cells rapidly expand after stem cell transplantation and are important for regulating GVHD and providing graft-versus-leukemia (GVL) effects. This review highlights the phenotypic and functional differences between UCB NK cells and adult peripheral blood (APB) NK cells, and discusses the possible therapeutic benefit of using UCB NK cells for adoptive immunotherapy in leukemia. Alloreactive NK cells show potent cytotoxic activities against human leukocyte antigen (HLA)-nonidentical leukemic cells and reduce leukemia relapses. The higher numbers of NK progenitors in UCB makes it a convenient source for ex vivo expansion of UCB NK cells for posttransplant treatment. UCB NK cells readily respond to interleukin-15, which may greatly enhance their antitumor effect. Activation and expansion protocols for UCB NK cells are currently being developed.