单元型相同骨髓移植后患者单核细胞来源的DC表面分子的检测

对单元型相同骨髓移植患者造血重建后的外周血单个核细胞加GM-CSF、IL-4进行DC诱导,7d后加入TNF-α于培养DC中,继续诱导3d。测定DC的表型、混合淋巴细胞反应对T细胞增殖能力的测定,并与健康志愿者外周血来源的DC进行比较。探讨单元型相同造血干细胞移植后患者单个核细胞来源的DC的生物学特性。结果显示,单元型相同造血干细胞移植患者外周血单个核细胞和正常人外周血单个核细胞来源的DC均高表达CD1α、CD83、CD80、CD86和HLA-DR等DC的相关抗原和共刺激分子,患者的未成熟DC经TNF-α诱导后,成为成熟和有功能的DC,单元型相同造血干细胞移植患者单个核细胞来源的DC在体外具有激发同种异体外周血T细胞增殖的能力,与健康人外周血来源DC组相比均无统计学意义(P>0.05)。     

脐带血中衍生树突状细胞及其生物学特性的研究

目的利用脐带血衍生培养树突状细胞(DC),以获得可用于临床治疗的一种新的血细胞制品.方法从脐带血中分离单个核细胞,联合使用细胞因子GM-CSF、IL-4和TNF-α在体外诱导和扩增DC;采用扫描电镜、流式细胞仪(FACS)和混合淋巴细胞反应(MLR)试验对脐带血来源的DC进行生物学特性分析.结果从30ml脐血诱导培养12d后可获得6.6×106的DC.扫描电镜观察具有典型的树突状突起;FACS分析显示,获得的DC细胞群高表达CD1a(90.6%)、CD80(96.8%)、CD86(84.8%)和HLA-DR(91.0%);MLR显示,脐带血诱导培养的DC对同种异体的脐带血幼稚型T细胞均具有强烈的激发和促增殖作用.结论从脐带血中成功诱导出大量高纯度成熟DC,为依赖DC免疫治疗的临床应用打下基础.     

脐带血中衍生树突状细胞及其生物学特性的研究

目的　利用脐带血衍生培养树突状细胞 (DC) ,以获得可用于临床治疗的一种新的血细胞制品 .方法　从脐带血中分离单个核细胞 ,联合使用细胞因子GM -CSF、IL - 4和TNF -α在体外诱导和扩增DC ;采用扫描电镜、流式细胞仪 (FACS)和混合淋巴细胞反应 (MLR)试验对脐带血来源的DC进行生物学特性分析 .结果　从 30ml脐血诱导培养 12d后可获得 6 .6× 10 6的DC .扫描电镜观察具有典型的树突状突起 ;FACS分析显示 ,获得的DC细胞群高表达CD1a(90 .6 % )、CD80 (96 .8% )、CD86(84 .8% )和HLA -DR(91.0 % ) ;MLR显示 ,脐带血诱导培养的DC对同种异体的脐带血幼稚型T细胞均具有强烈的激发和促增殖作用 .结论　从脐带血中成功诱导出大量高纯度成熟DC ,为依赖DC免疫治疗的临床应用打下基础     

钙离子载体体外诱导外周血造血干细胞定向分化为成熟树突状细胞的研究

目的以实体瘤患者外周血造血干细胞为来源，探讨体外诱导更成熟、功能更强的树突状细胞(DC)的方法。方法以血细胞分离机分离经动员的外周血造血干细胞(PBSC)，加入rhGM-CSF、rhIL-4和ma培养9d后分两组，其中一组洗去含细胞因子的培养液，并给予钙离子载体(CI)A23187处理24h，而另一组仍维持原培养液培养24h；对所获得的两组细胞分别进行细胞形态学、细胞表面抗原及刺激同种异体混合淋巴细胞反应(allo-MLR)能力的分析。结果两组细胞在倒置显微镜和扫描电镜下均显示典型的树突状细胞形态，流式细胞仪分析均高表达CD1α、HLA-DR、CD80和CD40分子；但给予A23187进一步处理24h的细胞比无A23187处理的细胞表达更丰富的CD86和CD83分子及具有更强的刺激allo-MLR的能力。结论组合细胞因子培养获得的PBSC来源的DC，经CI进一步诱导后可获得更成熟及功能更强的DC。     

氢化可的松对人外周血来源树突状细胞的诱导凋亡作用研究

旨在探讨氢化可的松对人外周血单核细胞来源树突状细胞 (DC )的凋亡诱导作用及其表型、功能的变化 ;采用细胞因子体外诱导人外周血来源DC ,加入不同浓度的氢化可的松进行培养 ;以细胞计数、PI染色测定细胞周期、间接荧光表型分析、ELISA法测定DC分泌的IL 12和3H TdR掺入测定DC对T细胞的激发作用 ;实验结果表明 ,外周血贴壁的单核细胞在细胞因子的诱导下可以分化为成熟的DC ,不同浓度 (2 μg/ml～ 5 0 μg/ml)的氢化可的松可下调DC表达的CD80和HLA DR分子 (分别下降 41 1%和 10 9% ) ,并使其分泌IL 12的能力明显下降 (由 38 1pg下降为 0 ) ,DC对自体T细胞的激发功能明显降低 ,而且一定浓度的氢化可的松还可以使DC发生凋亡 (凋亡率可达 5 6 6 % )。糖皮质激素不仅通过下调DC表达的协同刺激分子及其分泌的IL 12从而影响DC对T细胞的激发作用 ,而且可以直接诱导DC发生凋亡 ,从而下调免疫应答的发生。     

不同大鼠胶质瘤抗原致敏的DC体外诱导CTL活性的研究

目的: 比较大鼠骨髓来源的树突状细胞(dendritic cells, DC)体外经由大鼠C6胶质瘤细胞由不同方式制备的不同抗原致敏后,对特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte, CTL)的诱导作用。方法: 自大鼠骨髓分离DC前体细胞,经重组大鼠粒细胞巨噬细胞集落刺激因子(rrGM-CSF)+白细胞介素4(rrIL-4)诱导培养、扩增;由C6胶质瘤细胞经由反复冻融、煮沸灭活及超声破碎细胞抽提其总蛋白的方法制备各种不同抗原致敏DC,致敏的DC与T淋巴细胞进行共培养诱导CTL;以ELISA法检测CTL诱导过程中淋巴细胞趋化因子(lymphocyte chemoattractant factor)及细胞因子IFN-γ分泌水平:以 -TdR掺入法检测DC诱导T细胞增殖及其特异性CTL杀伤活性。 结果: 体外应用煮沸灭活瘤细胞制备的肿瘤抗原致敏DC,能诱导更强的刺激T细胞增殖的能力、并且可以诱导杀伤活性更强的CTL。结论: 应用煮沸灭活的瘤细胞制备瘤抗原负载DC获得瘤苗可获得更强的抗肿瘤保护作用。     

趋化性细胞因子MIP-1α促小鼠外周血DC前体细胞动员的实验研究

抗MIP-1α抗体可以阻断P.acnes对DC前体细胞的动员作用。对C57BL/6J(B6)小鼠静脉直接注射MIP-1α,24h后在B6小鼠外周血中F4/80-B220-CD11c+细胞明显增多,占外周血单个核细胞(PBMC)13.47%±1.4%。新鲜分离的F4/80-B220-CD11c+细胞不具有成熟DC的特征,经过细胞因子GM-CSF、IL-4和mTNF-α体外培养7d后的F4/80-B220-CD11c+细胞呈现树突状突起并形成细胞团簇,高度表达Ia、CD11c、DEC205、CD80、CD86,中度表达CD40,不表达CD8α、F4/80表面标志,并具有极强的刺激异源性T细胞增殖的能力。总之,研究表明趋化性细胞因子MIP-1α参与调节DC前体细胞的动员,并且注射MIP-1α可以直接迅速动员F4/80-B220-CD11c+DC前体细胞进入小鼠外周血。实验首次提出了用趋化性细胞因子MIP-1α动员DC前体细胞进入外周血的思路和实践,为体外获取大量功能正常的DC开辟了新的便捷途径。     

腺病毒介导HSP70-HBsAg嵌合基因转染树突状细胞及生物学特性分析

目的 分析携带热休克蛋白70(HSP70)和乙肝表面抗原(HBsAg)嵌合基因的腺病毒表达载体感染人外周血诱导培养树突状细胞(DC)后其生物学特性的改变.方法 从正常人外周血中分离单个核细胞,经Ad-HSP70-HBsAg感染刺激后.在含细胞因子粒细胞巨噬细胞集落刺激因子(GM-CSF)、白介素-4(IL-4)、肿瘤坏死因子(TNF-α)的培养液体中体外诱导培养DC;采用荧光倒置显微镜、RT-PCR、流式细胞仪(FACS)和混合淋巴细胞反应(MLR)试验对人外周血来源的DC进行生物学特性分析.结果 Ad-HSP70-HBsAg感染后的DC能够有效表达示踪蛋白GFP和转录基因HSP70-HBsAg,病毒感染组和对照组的DC在细胞形态、DC细胞表面分子表达和刺激同种异体的人外周血幼稚型T细胞增殖能力的方面无明显差别.结论 Ad-HSP70-HBsAg可有效转染DC,感染后的病毒对DC的生长和生物学特性无显著影响.     

树突状细胞成熟及其信号传导基因表达谱的研究

目的利用多种细胞因子及肿瘤细胞裂解物刺激诱导树突状细胞(dendritic cell,DC)成熟,并利用基因芯片技术对其信号传导基因表达谱进行研究。方法外周血单个核细胞在体外用细胞因子GM-CSF(100 ng/m l),IL-4(1 000 U/m l)诱导下获取DC,以乳腺癌MCF-7细胞反复冻融的裂解物冲击DC,通过形态学和FCM检测来确定DC。再用基因芯片技术检测其细胞因子及信号传导相关基因表达的变化。结果DC在细胞因子的诱导下,形态上伸出许多伪足样突起,在摄取抗原后可见内吞样小泡。FCM检测呈现成熟DC的标志:CD40、CD83、CD80、CD86、HLA-DR等高表达。芯片扫描发现16种信号传导因子发生5倍以上的改变,其中包括钙调磷酸酶结合蛋白1、TGF-β3受体、FKBP-雷帕霉素相关蛋白、小分子细胞因子A(Cys-Cys)、人T细胞特异蛋白(RANTES)等。结论通过体外细胞因子诱导及抗原冲击的成熟DC不但在形态和表型上存在明显差异,而且在功能和信号传导方面也存在显著变化。     

γδ T细胞诱导与分化的初步研究-Ⅰ

目的:探讨在异戊烯焦磷酸(IPP)作用下,健康人外周血和新生儿脐带血中γδT细胞的增殖和亚型变化,以期获得足够的具有不同特征的γδT细胞用于后续的实验研究。方法:流式细胞术分析经IPP诱导的单个核细胞表面分子的表达状态,评估其γδT细胞的比例、亚型和表型格局。结果:健康人外周血和新生儿脐带血中γδT细胞数量虽占较低比例,但两者的γδT细胞存在显著异质性。健康人外周血γδT细胞主要以Vγ9Vδ2 TCR亚群为主;初始分选的外周血单个核细胞中Vγ9Vδ2T细胞主要为中央记忆型(CD27+CD45RA-)和效应记忆型(CD27-CD45RA-);IPP诱导后,γδT细胞显著扩增,Vγ9Vδ2 T细胞亚群转变为以效应记忆型为主,并高表达HLA-DR和B7分子。而新生儿脐带血来源的γδT细胞却呈现出较为复杂的亚群异质性,其Vγ9Vδ2 T细胞主要为CD27+CD45RA+的幼稚型细胞;经IPP诱导14天后,γδT细胞获得扩增(比例增高)且Vγ9Vδ2 T细胞趋向中央记忆型和效应记忆型分化,但仍以幼稚型为主。结论:健康人外周血和新生儿脐带血中γδT细胞在数量、亚群诸方面存在差异;IPP具有诱导和扩增外周血γδT细胞的生物学作用,而脐带血来源的γδT细胞还需相关细胞因子的协同作用,才能呈现出其分化成熟为可用于理论研究和临床实验的效应记忆型γδT细胞的潜能。关于其具有的免疫调节和效应功能将另文报道。     

人外周血及脐血来源树突状细胞对宫颈癌细胞的直接杀伤效应

探讨人外周血及脐血来源树突状细胞(DC)的生物学特性及其对宫颈癌细胞的直接杀伤效应。自外周血及脐血分离获得单核细胞,体外经rhGM-CSF、rhIL-4和rhTNF-α诱导培养,获得DC,采用流式细胞仪(FCM)检测DC相关表型;MTT法检测人外周血及脐血来源DC对宫颈癌细胞HeLa及CaSki的体外直接杀伤效应。结果显示,人外周血及脐血来源单核细胞诱导培养第6天后具有典型DC形态,FCM结果显示高表达HLA-DR(分别为99.29%和98.14%)、CD86(分别为99.45%和90.92%)和CD209(分别为87.44%和83.14%),而CD14表达率较低(分别为0%和3.32%);抑瘤实验显示人外周血及脐血来源DC对宫颈癌细胞HeLa及CaSki均有明显杀伤效应。研究提示,人外周血及脐血来源DC对人宫颈癌细胞具有直接杀伤效应,两者具有相似的生物学特性,为DC在肿瘤免疫治疗中的作用提供了新思路。     

A Cryopreservation Method of Human Peripheral Blood Mononuclear Cells for Efficient Production of Dendritic Cells

The establishment of a cryopreservation method for unstimulated fresh peripheral blood mononuclear cells (PBMC) with nearly 100% viability would greatly contribute to the conduct of various immunological experiments. The cells most sensitive to freezing and thawing procedure seem to be dendritic cells (DC) and their precursors, which are of the most potent antigen-presenting cells. The authors investigated and established a method of cryopreserving fresh PBMC from which DC were recovered and differentiated efficiently by using recombinant (r) GM-CSF and rIL-4. PBMC frozen in the presence of 12% dimethylsulfoxide and 25–30% fetal calf serum recovered DC as efficiently as freshly obtained PBMC. Established DC could also be cryopreserved in the presence of 12% DMSO with their viability maintained at more than 90%. The 12% DMSO freezing solutions were superior to both the 10% DMSO solution and the previously reported DC freezing medium (2 m or 15.4% DMSO). The DC obtained from the cryopreserved PBMC expressed HLA-DR, HLA-DQ, CD80 and CD86 antigens, and stimulated allogenic PBMC to an extent almost identical to that obtained from fresh PBMC. These findings indicate that the conditioned medium utilized here enables safe cryopreservation of DC and DC precursors in PBMC.     

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。     

人外周血CD4~+树突状细胞的纯化及鉴定

目的　建立人外周血树突状细胞 (dendriticcell,DC)的分离方法 ,观察其形态学和免疫组织化学特点 ,为下一步细胞融合提供DC来源。方法　以免疫磁珠分选法从人外周血单个核细胞中分离CD4 + DC ,流式细胞仪检测所得细胞的纯度 ,光镜、电镜和激光共聚焦扫描显微镜观察其形态 ,SP免疫细胞化学方法检测DC的分子表达。结果　此纯化方法所得细胞纯度可达到 80 %以上 ,形态学观察可见纯化细胞具有典型的DC特征 ,该细胞能高表达HLA DR和S 10 0分子。结论　免疫磁法可获得较高纯度典型DC ,为进一步进行DC与肿瘤的融合实验及临床应用提供了可能     

Dendritic Cells Generated from Human Blood in Granulocyte Macrophage-Colony Stimulating Factor and Interleukin-7

Dendritic cells (DC), with potentially important clinical applications, have been generated from human peripheral blood monocytes in the presence of GM-CSF and IL-4 (G4 DC). In the present report we show that DC with a novel phenotype can be generated from blood adherent mononuclear cells in the presence of GM-CSF and IL-7 (G7 DC).

Adherent cells from PBMC, cultured in GM-CSF (600 U/ml) and IL-7 (6 U/ml), were transformed over 7 days into cells with DC morphology, at a yield of 1.2 − 1.6 × 10⁶ per 10⁷ PBMC. G7 DC not only expressed class I and class II MHC, CD1a, CD11c, CD23, CD40, CD54, CD58, CD80, CD86 and CD95, like G4 DC, but also CD21, which is the complement receptor type 2, a ligand for CD23 and a receptor for EBV and IFN-.

G7 DC were at least one log more effective in the autologous MLR and at least two logs more effective in the allogeneic MLR, than PBMC. They elicited proliferative responses of CD4 T cells to tetanus toxoid and CD8 T cells to an EBV peptide, and stronger T-cell cytotoxicity to EBV peptide than G4 DC. Expression of CD21 by G7 DC suggests that IL-7 delivers a distinct signal to DC precursors and that G7 DC may be functionally distinct.     

激活的脐血树突状细胞对肿瘤细胞的直接杀伤作用

探索脐血树突状细胞 (DC)在γ干扰素 (IFN γ )及细菌脂多糖 (LPS )激活前后对肿瘤细胞杀伤活性的差异。分离健康脐血单核细胞 ,用重组粒单细胞集落刺激因子 (GM CSF )、白介素 4 (IL 4 )和α肿瘤坏死因子α (TNF α )诱导为DC。于诱导第11天在合成培养基中加入LPS或IFN γ继续培养 12h ,将其激活。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Jurkat及Daudi为靶细胞 ,以不同效靶比 (E∶T )与DC共同培养 18h ,采用51Cr释放试验检测DC激活前后抗肿瘤活性的差异。结果 (1)LPS及IFN γ可不同程度地上调DC表面CD86、CD80、CD83及CD1a的表达 ,尤以LPS刺激组明显 ;(2 )DC在未加刺激因子前能有效杀伤Jurkat细胞 ,在效靶比为 2 0∶1时 ,LPS或IFN γ可使其杀伤活性进一步提高至 5 0 0 %、 36 9% ,均与未加刺激因子前有显著差异 (P <0 0 0 1,P <0 0 2 5 ) ;(3)在效靶比为 2 0∶1时 ,LPS可使DC对Daudi的杀伤率提高至 19 8% (P <0 0 2 5 ) ,而未加刺激因子组及IFN γ刺激组DC对Daudi在任何效靶比均未显示明显的杀伤作用。LPS或IFN γ激活的脐血DC对Jurkat及Daudi细胞具有选择性杀伤作用     

Interleukin 1 Activity Produced by Human Rheumatoid and Normal Dendritic Cells

Dendritic cells (DC) from the synovial inflammatory tissue and peripheral blood of patients with rheumatoid arthritis and from the peripheral blood of normal blood donors were compared with the autologous monocytes for their capacity to produce and release interleukin 1 (IL-1). Synovial DC often spontaneously released higher amounts of IL-1 activity than unstimulated and lipopolysaccharide-stimulated peripheral blood DC and monocytes. The IL-1 production by both DC and monocytes increased after stimulation with bacterial lipopolysaccharide. In contrast with synovial DC the peripheral blood DC from both patients with rheumatoid arthritis and normal controls released less IL-1 activity than peripheral blood monocytes did. Inhibition with an antiserum to IL-1 revealed that IL-1 production is important for the accessory activity of the peripheral blood DC. Thus human DC from inflammatory sites and peripheral blood produce IL-1 activity.     

Mesenchymal Stem Cells Inhibit Dendritic Cell Maturation and Their Allosti mulatory Capacity

1 IntroductionDendritic cells (DC) are the most potent antigen-presenting cells. They play an important role in both initiation of immunity and maintenance of immune tolerance. In the recent years, they have been used in humans for the treatment of tumors. DCs are very poor in blood; however, they can be generated in vitro from either CD34~ hematopoietic stem cell precursors or peripheral blood monocytes, by using appropriate cytokines~([1]). However, the microenvironment can influence their differentiatio...     

Function and subsets of dendritic cells and natural killer cells were decreased in gastric cancer

Dendritic cells (DCs) and natural killer (NK) cells initiate specific immune responses against tumor cells. The aim of the present study was to determine the cytotoxicity and the subsets of the DC and NK cells and the cytokines level of DC and NK cells from cancer tissue and peripheral blood in the gastric cancer patients. Cytotoxicity of DC and NK was determined using the Cytotox non-radioactive assay. The cytotoxic activity of DC or NK isolated from cancer tissue and peripheral blood was attenuated in gastric cancer patients. CD11c, CD80, CD83, CD16, CD57 and CD69 were decreased in the cancer tissue and peripheral blood in the gastric cancer patients. CD86, CCR7 and CD59 were no significance in the cancer tissue and peripheral blood from gastric cancer patients. Tumor necrosis factor (TNF)-α, interleukin (IL)-2, T-bet and IL-15Rβ levels were decreased in DC and NK from the gastric cancer tissue and peripheral blood in the gastric cancer patients. IL-15 and IL-15Rα level were no significance in DC and NK in the gastric cancer tissue and peripheral blood in the gastric cancer patients. These results indicate that the cytotoxic activity and subsets and cytokines of DC and NK cells in the cancer tissue and peripheral blood in the gastric cancer patients were decreased. The decrease of subsets content and cytokines of DC and NK may contribute to a decrease in the function of DC and NK in the tissue and peripheral blood in the gastric cancer patients.     

Dendritic Cells and Monocytes as Accessory Cells in T-Cell Responses in Man

A method is described for simple and rapid preparation of human dendritic cells (DC) and monocytes (Mo) from peripheral blood. The phenotype of enriched DC and Mo was determined and compared by means of a panel of monoclonal antibodies (Mab's). The distribution and quantitative expression of HLA class II molecules encoded by the subloci DP, DQ, and DR were the same on the two cell types. During in vitro culture a rapid decrease of class II antigens on Mo was observed, whereas the expression of class II antigens on DC was relatively stable. The absence of monocyte markers on DC may indicate that this cell type does not belong to the monocyte/macrophage cell lineage. The phenotypic analysis shows that peripheral blood DC also lack differentiation antigens expressed by epidermal Langerhans cells (OKT6) and lymph node follicular dendritic reticulum cells (DRC-1). The relationship between peripheral blood DC and tissue-localized DC thus remains unsolved. With relatively high numbers of DC now available, production of DC lineage-specific Mab's may be approached.