In vitro generation of dendritic cells from human blood monocytes in experimental conditions compatible for in vivo cell therapy

DC are professional APC that are promising adjuvants for clinical immunotherapy. Methods to generate in vitro large numbers of functional human DC using either peripheral blood monocytes or CD34+ pluripotent HPC have been developed recently. However, the various steps of their in vitro production for further clinical use need to fit good manufacturing practice (GMP) conditions. Our study focused on setting up such a full procedure, including collection of mononuclear cells (MNC) by apheresis, separation of monocytes by elutriation, and culture of monocytes with GM-CSF + IL-13 + autologous serum (SAuto) in sterile Teflon bags. The procedure was first developed with apheresis products from 7 healthy donors. Its clinical feasibility was then tested on 7 patients with breast cancer. The characteristics of monocyte-derived DC grown with SAuto (or in some instances with a pooled AB serum) were compared with those obtained in the presence of FBS by evaluation of their phenotype, their morphology in confocal microscopy, and their capacity to phagocytize latex particles and to stimulate allogeneic (MLR) or autologous lymphocytes (antigen-presentation tests). The results obtained demonstrate that the experimental conditions we set up are easily applicable in clinical trials and lead to large numbers of well-defined SAuto-derived DC as efficient as those derived with FBS.     

健康人外周血树突状细胞体外培养及表型鉴定

目的 分离人外周血单个核细胞,经体外诱导培养获取成熟树突状细胞(DC),并对树突状细胞表型表达鉴定,为进一步研究DC功能提供基础.方法 取健康成人新鲜外周血,经密度梯度离心法分离获得外周血单个核细胞,2 h贴壁后加入粒细胞集落刺激因子(GM-CSF)、白介素(IL)-4,第6天加入肿瘤坏死因子-α(TNF-α)刺激DC成熟,倒置显微镜观察每日细胞形态;分别于第1天、第6天、第8天用流式细胞仪对其进行表型鉴定;同种异体混合淋巴细胞反应观察对T细胞的抗原提呈能力 倒置显微镜下DC细胞形态不规则,表面有毛刺状突起,呈DC典型形态学特征;成熟DC细胞表面CD1a、CD80、CD83、人类白细胞抗原(HLA)-DR表达明显增加,混合淋巴细胞反应OD值增加.结论 用GM-CSF、IL-4和TNF-α可以诱导健康成人外周血单个核细胞,培养出成熟的DC细胞,为进一步研究DC功能提供基础.     

体外纯化培养外周血来源的树突状细胞的研究

目的建立来源于外周血的树突状细胞(DCs)的纯化培养方法并观察DCs的形态及功能。方法以Fi-coll密度梯度离心法从正常人外周血中分离出外周血单个核细胞(PBMNC)后,用体外培养的手段,采用培养黏附法或经免疫磁珠筛选法,获得单核细胞;分别加入不同浓度的细胞因子(重组人粒细胞-巨噬细胞集落刺激因子重组人白介素4重组人肿瘤坏死因子α)诱导培养,培养12 d诱导出DCs;用光镜和电镜观察培养的DCs,用流式细胞仪检测DCs表面的细胞表型(CD80、CD83、CD86、CD1α、HLA-DR),MTT法检测DC对T细胞的刺激增殖效应。结果在体外诱导出外周血来源的成熟DCs,电镜和光镜分析表明具有DCs的典型形态,所培养的细胞表面高表达CD80、CD83、CD86、CD1α、HLA-DR,并具有刺激异基因淋巴细胞增殖的能力。结论应用外周血来源的单个核细胞,采用培养黏附法或免疫磁珠分选法分选出的单核细胞,细胞因子培养12 d可得到成熟正常的DCs。     

人外周血单核细胞体外诱导树突状细胞的形态学观察与功能研究

目的：探讨从人外周血单核细胞体外培养扩增的树突状细胞(dendritic cell,DC)形态学特征及其活性。方法：人外周血常规分离单个核细胞，粘附6h后去除悬浮细胞，以细胞因子粒细胞-巨噬细胞集落刺激因子(GM-CSF)、白细胞介素-4(IL-4)进行诱导培养7d，并进行形态学特征，细胞表型和淋巴细胞刺激能力鉴定。结果：培养1周即可得到大量DC，形态学观察可见细胞形态不规则，细胞表面大量突起，为典型的DC特征，免疫组化和间接免疫荧光显示CDla阳性表达率达80％～95％，并能刺激同种淋巴细胞增殖反应。结论：人外周血单核细胞可经GM-CSF、IL-4诱导培养成DC，具有典型的树突状细胞的形态学特征及抗原呈递能力。     

人外周血单核细胞来源的树突状细胞的体外诱导

目的:探讨在体外从人外周血单核细胞诱导培养成熟的树突状细胞(dendritic cell,DC)的方法.方法:培养过程分两阶段,第一阶段:采用密度梯度离心法分离健康成人外周血中的单个核细胞,再以黏附法分离出单核细胞,经重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)100 ng/mL、重组人白介素-4(rhIL-4)100 ng/mL体外诱导.第二阶段:第5天加入重组人肿瘤坏死因α-(rhTNF-α)100 ng/mL,继续培养2 d,刺激DC成熟.倒置显微镜下观察DC形态,流式细胞仪检测DC表面标志物CD83、CD1a、CD86、CD40、CD14表达水平,用MTT法测定DC刺激同种异体T细胞增殖的能力.结果:人外周血单核细胞经rhGM-CSF及rhIL-4诱导培养5 d后,多数细胞呈集落生长,细胞表型CD83、CD1a、CD86、CD40及CD14分别是14.3%、12.8%、20.1%、19.9%及16.2%.加入rhTNF-α诱导后,即培养第7天,细胞表型CD83、CD1a、CD86、CD40及CD14分别是29.8%、18.2%、33.6%、28.1%及8.0%(与第5天比较,均P<0.05).成熟后的DC具备较强刺激T细胞增殖的能力.结论:rhGM-CSF联合rhlL-4诱导人外周血单核细胞,可获得大量不成熟的DC,该体系有利于Dc扩增,加入rhTNF-α,继续培养2 d,可诱导出成熟的DC,成熟的DC具备较强刺激T细胞增殖的能力.     

明胶法富集外周血单核细胞并刺激其成熟为树突状细胞

背景：如何简易高效分离纯化人外周血单核细胞并刺激成熟为树突状细胞,未见标准化操作流程。目的：观察明胶法分离外周血单核细胞的效率以及将分离出的单核细胞刺激成熟为树突状细胞的表型特征,并与普通塑料黏附法对比。方法：使用人淋巴细胞分离液分离人外周血得到单个核细胞,根据培养瓶是否进行明胶包被分为明胶包被组和普通塑料组。均分单个核细胞,按组别分离获得单核细胞并诱导刺激成熟为树突状细胞。计数各组所得单核细胞数,使用流式细胞仪检测2组单核细胞的CD14阳性率、T、B淋巴细胞污染率、树突状细胞非成熟期和成熟期CD1a,CD83的表达情况,锥虫蓝拒染法计算细胞活率,观察对比2组血小板污染情况。结果与结论：明胶包被组单核细胞数及CD14阳性率显著高于普通塑料组（P〈0.05）,普通塑料组淋巴细胞污染率显著高于明胶包被组（P〈0.05）。2组细胞活率及树突状细胞表型差异无显著性意义（P〉0.05）。明胶包被组血小板污染率低于普通塑料组。提示明胶法可以简单高效分离出单核细胞并成功刺激成熟为树突状细胞。     

In vitro generation of antigen-specific hemolytic plaque-forming cells from human peripheral blood mononuclear cells

We have described a culture and assay system for the sensitization of human peripheral blood mononuclear cells with a T cell-dependent antigen, sheep erythrocytes, in the absence of nonspecific stimulatory agents and with the subsequent generation of macroscopic hemolytic plaques. We have shown that the antibody produced by the plaque-forming cells generated in this culture system is specific for the sensitizing antigen, and that the plaques created are not false plaques because their formation is inhibited by cycloheximide. The success of this system can be attributed to several critical factors including large numbers of peripheral blood mononuclear cells (5 x 10(6) culture), a prolonged period of incubation (10-11 d), continuous rocking during the entire period of incubation, culturing in large (35-mm) flat-bottomed culture dishes in the presence of human plasma, and the appropriate antigen concentration (5 x 10(6) sheep erythrocytes/culture). Furthermore, the generation of macroscopic hemolytic plaques requires plaquing sensitized peripheral blood mononuclear cells in target cell monolayers fixed in an agarose matrix with an incubation period of 2-3 h. We have further shown that the antigen-specific response measured by this system is dependent on adherent cells and T lymphocytes. At least one population of the helper T cells is sensitive to 2,000 rad irradiation. This system is simple, sensitive, and should serve as an effective tool for the analysis of cellular interactions involved in the generation of human antigen-specific plaque-forming cells, the genetic control the human immune response, and the pathophysiology of altered immunoregulation in disease.     

脐血CD34+细胞和外周血单核细胞两种来源的树突状细胞特性的比较研究

目的　体外大量扩增和纯化具有典型表型、形态和功能的树突状细胞（ＤＣ）,以进行相关基础研究和临床应用。方法　采用免疫磁珠法分离脐血ＣＤ３４ ＋ 细胞及外周血去Ｂ、去Ｔ淋巴细胞的单个核细胞（ 单核细胞） ,然后以ＧＭＣＳＦ、ＩＬ４、ＴＮＦα、Ｆｌｔ３ 配基（ＦＬ）、ＳＣＦ等不同的细胞因子配伍,分别诱生ＤＣ,通过流式细胞仪、电镜、光镜分析其特性,同时检测其刺激同种Ｔ细胞增殖的能力。结果　脐血与外周血诱生ＤＣ的方案不同,由脐血ＣＤ３４＋ 细胞诱导ＤＣ 时,ＧＭＣＳＦ＋ ＴＮＦα＋ ＳＣＦ＋ ＦＬ 组合可使ＣＤ１ａ＋ 细胞比例增至（２７ ．１８ ±１－５６）％ ,明显高于单独应用ＧＭＣＳＦ组［（０．６５±０ ．３８）％ ］ 。外周血单核细胞诱导的ＤＣ,则ＧＭＣＳＦ＋ 高剂量ＩＬ４（１０００ Ｕ／ｍｌ） 组合效率最高,诱生的ＣＤ１ａ ＋ 细胞可达（２１ ．８０ ±０－３２） ％ 。两种来源的ＤＣ在表型及形态上差异无显著性,两者同样具有刺激同种异体淋巴细胞增殖的能力。结论　从脐血和外周血均可诱生ＤＣ,根据应用目的的不同对其进行选择,这为ＤＣ用于临床治疗选择不同细胞来源提供了实验基础。     

人外周血树突状细胞的体外诱导培养

目的研究rhGM-CSF和rhIL-4培养体系对树突状细胞(DendriticCell,DC)的诱导培养。方法分离正常人或多发性骨髓瘤(MM)患者外周血中单个核细胞,体外培养树突状细胞。观察培养的DC的形态变化,并应用流式细胞仪分析DC表面的免疫分子的表达。结果100ng/mlrhGM-CSF和500U/mlrhIL-4能使正常人或多发性骨髓瘤患者外周血中单个核细胞分化发育为高表达CD80、CD86、HLAⅠ类和HLAⅡ类抗原的DC。结论rhGM-CSF和rhIL-4能使树突状细胞定向分化成为抗原提呈细胞,为DC疫苗治疗肿瘤感染性疾病奠定了基础。     

In vitro clinical-grade generation of red blood cells from human umbilical cord blood CD34+ cells

BACKGROUND: There is no appropriate alternative source of red blood cells (RBCs) to relieve the worsening shortage of blood available for transfusion. Therefore, in vitro generation of clinically available RBCs from hematopoietic stem cells could be a promising new source to supplement the blood supply. However, there have been few studies about the generation of clinical‐grade RBCs by coculture on human mesenchymal stem cells (MSCs) and various cytokine supplements, even though the production of pure RBCs requires coculture on stromal cells and proper cytokine supplements. STUDY DESIGN AND METHODS: Umbilical cord blood (CB) CD34+ cells were cultured in serum‐free medium supplemented with two cytokine sets of stem cell factor (SCF) plus interleukin‐3 (IL‐3) plus erythropoietin (EPO) and SCF plus IL‐3 plus EPO plus thrombopoietin (TPO) plus Flt‐3 for 1 week, followed by coculture upon MSCs derived from bone marrow (BM) or CB for 2 weeks. RESULTS: Almost pure clinical‐grade RBCs could be generated by coculturing with CB‐MSCs but not BM‐MSCs. Expansion fold and enucleation rate were significantly higher in coculture with CB‐MSCs than BM‐MSCs. Despite a 2.5‐fold expansion of erythroblasts in the presence of TPO and Flt‐3 for 8 days, the final RBC count was higher without TPO and Flt‐3. CONCLUSIONS: This study is the first report on generating clinical‐grade RBCs by in vitro culture with human MSCs and compared effectiveness of several cytokines for RBC production. This provides a useful basis for future production of clinically available RBCs and a model of erythropoiesis that is analogous to the in vivo system.     

人外周血树突状细胞的体外诱导培养

目的研究rhGM-CSF和rhIL-4培养体系对树突状细胞(Dendritie Cell,DC)的诱导培养.方法分离正常人或多发性骨髓瘤(MM)患者外周血中单个核细胞,体外培养树突状细胞.观察培养的DC的形态变化,并应用流式细胞仪分析DC表面的免疫分子的表达.结果100ng/ml rhGM-CSF和500U/ml rhIL-4能使正常人或多发性骨髓瘤患者外周血中单个核细胞分化发育为高表达CD80、CD86、HLAⅠ类和HLAⅡ类抗原的DC.结论rhGM-CSF和rhIL-4能使树突状细胞定向分化成为抗原提呈细胞,为DC疫苗治疗肿瘤感染性疾病奠定了基础.     

Effect of ex vivo culture duration on phenotype and cytokine production by mature dendritic cells derived from peripheral blood monocytes

BACKGROUND: To generate clinical-grade dendritic cells (DCs) ex vivo for immunotherapy trials, peripheral blood monocytes are typically cultured in granulocyte-macrophage–colony-stimulating factor (GM-CSF) and interleukin (IL)-4 and then matured using one or more agents. Duration of the initial DC culture is one important variable that has not been systematically evaluated for its effect on the characteristics of the final mature DC product.
STUDY DESIGN: DCs were generated from elutriated peripheral blood monocytes by incubation in medium containing 2000 units per mL each of GM-CSF and IL-4 for 3 to 7 days, followed by maturation with lipopolysaccharide and interferon-γ (IFN-γ). DC yield, viability, flow cytometric phenotype, and cytokine production were evaluated.
RESULTS: The percentage yield and viability of mature DCs were similar after GM-CSF/IL-4 culture for 3 or 7 days. In either case, mature DCs expressed abundant CD80, CD86, CD83, and CCR7, but 3-day DCs expressed these antigens in a more consistent and homogeneous manner. Mature 3-day DCs produced much more IL-12 and less IL-10 after restimulation with CD40L-LTK than 7-day DCs. The former were also more effective in presenting immunogenic peptides to CD8 T cells. Analogous changes in cytokine production were observed in mature DCs prepared using lower concentrations of GM-CSF/IL-4 or when the alternative maturation cocktails poly(I:C)/IFN-γ and soluble CD40L/IFN-γ were used.
CONCLUSION: Extended initial culture of DCs in GM-CSF/IL-4 does not affect yield or viability of subsequently matured DCs, but can adversely affect their ability to homogeneously express high levels of functionally important surface molecules such as CD83 and CCR7 and to produce IL-12.     

脐血CD34^＋细胞和外周血单核细胞两种来源的树突状细胞特性？…

目的 体外大量扩增和纯化具有典型表型、形态和功能的树突状细胞（ＤＣ）、以进行相关基础研究和临床应用。方法 采用免疫磁珠江分离脐血ＣＤ３４＾＋细胞及外周血去Ｂ、去Ｔ淋巴细胞的单个核细胞（单核细胞）,然后以ＧＭ－ＣＳＦ、ＩＬ－４、ＴＮＦα、Ｆｌｔ３配基（ＦＬ）、ＳＣＦ等不同的细胞因子配伍分别诱生ＤＣ,通过流式细胞仪、电镜、光镜分析其特性,同时检测其刺激同种Ｔ细胞增殖的能力。结果 脐民外周血诱生ＤＣ的方     

成人外周血平滑肌祖细胞的体外培养

背景：外周血平滑肌祖细胞具有向平滑肌细胞分化的能力。目的：探索体外分离培养成人外周血平滑肌祖细胞的方法及其生长分化特性。方法：采用密度梯度离心法分离获得成人外周血单个核细胞,培养12d后,应用流式细胞仪鉴定分析平滑肌祖细胞并分选纯化,继续培养诱导分化。采用倒置显微镜观察平滑肌祖细胞的形态变化,免疫荧光染色法观察其α-肌动蛋白的表达,同时应用Westernblot法检测调宁蛋白、平滑肌肌球蛋白重链的表达情况。此外,观察平滑肌祖细胞的生长特性,绘制生长曲线。结果与结论：成人外周血单个核细胞诱导培养4d时开始出现细胞集落,12d时细胞呈明显梭形。流式细胞仪分析显示,CD14和CD105双阳性的平滑肌祖细胞占贴壁细胞的（71.8±7.2）%。分选纯化的平滑肌祖细胞培养到28d呈旋涡状生长。间接免疫荧光染色显示,α-肌动蛋白表达呈阳性。Westernblot检测显示,调宁蛋白和平滑肌肌球蛋白重链分别于14,21d开始表达,并逐渐增加。生长曲线表明,细胞生长至第6天进入对数生长期,第12天后进入平台期。提示通过对外周血单个核细胞的诱导培养,可以获得大量的平滑肌祖细胞。它能稳定增殖,并可进一步分化为平滑肌细胞。     

成人外周血平滑肌祖细胞的体外培养

背景:外周血平滑肌祖细胞具有向平滑肌细胞分化的能力.目的:探索体外分离培养成人外周血平滑肌祖细胞的方法及其生长分化特性.方法:采用密度梯度离心法分离获得成人外周血单个核细胞,培养12 d后,应用流式细胞仪鉴定分析平滑肌祖细胞并分选纯化,继续培养诱导分化.采用倒置显微镜观察平滑肌祖细胞的形态变化,免疫荧光染色法观察其α-肌动蛋白的表达,同时应用Western blot法检测调宁蛋白、平滑肌肌球蛋白重链的表达情况.此外,观察平滑肌祖细胞的生长特性,绘制生长曲线.结果与结论:成人外周血单个核细胞诱导培养4 d时开始出现细胞集落,12 d时细胞呈明显梭形.流式细胞仪分析显示,CD14和CD105双阳性的平滑肌祖细胞占贴壁细胞的(71.8±7.2)%.分选纯化的平滑肌祖细胞培养到28 d呈旋涡状生长.间接免疫荧光染色显示,α-肌动蛋白表达呈阳性.Western blot检测显示,调宁蛋白和平滑肌肌球蛋白重链分别于14,21 d开始表达,并逐渐增加.生长曲线表明,细胞生长至第6天进入对数生长期,第12天后进入平台期.提示通过对外周血单个核细胞的诱导培养,可以获得大量的平滑肌祖细胞.它能稳定增殖,并可进一步分化为平滑肌细胞.     

人外周血树突状细胞的分离与鉴定

目的:体外分离培养并鉴定人外周血树突状细胞,并观察其抗原呈递功能。方法:实验于2005-05/2006-11在南方医科大学南方医院肿瘤中心生物治疗实验室完成。从人类白细胞抗原A2表达阳性的健康人外周血中分离获得单个核细胞。培养5h后洗涤贴壁细胞,加入含有10%人AB血清的RPMI1640培养基,及重组人粒细胞-巨噬细胞集落刺激因子和重组人白细胞介素4,于培养的第1,3,6天对树突状细胞的形态、表型进行分析,并定期检测树突状细胞的纯度与得率。抽取与以上树突状细胞不同来源的其他健康人外周血。经淋巴细胞分离液分离后,获取非贴壁细胞,用含10%人AB血清的1640培养基重悬,加入白细胞介素2继续孵育6d,作为同种异体T淋巴细胞。将树突状细胞分为两组,一组按常规方法培养6d,另一组在培养至第5天时加入黑色素瘤抗原基因A3编码的多肽继续培养24h。在经紫外线处理后的96孔板中,分别加入树突状细胞悬液1×104,5×103,2×103,1×103细胞/每孔,以自身T淋巴细胞作为对照,每孔设3个复孔,分别加入1×105淋巴细胞/每孔。评价树突状细胞刺激T淋巴细胞增殖的能力。结果:①单个核细胞体外培养至第6天,可获得大量、90.81%高纯度的树突状细胞,能够较高地表达21.8?1a、99.0%HLA-DR、63.4?80、18.9?83和80.6?86。②将诱导培养6d获得的两组树突状细胞作为刺激细胞,以不同的浓度与同种异体淋巴细胞混合,均可产生增殖反应;经过黑色素瘤抗原基因A3编码的多肽处理的各种比例的树突状细胞,较相应未经黑色素瘤抗原基因A3编码的多肽处理的树突状细胞激发淋巴细胞增殖的能力明显增强,浓度相对较高的树突状细胞刺激效果最明显,能够强烈地激发同种混合淋巴细胞增殖。结论:得到了一群较高程度表达CD83、CD86和HLA-DR分子、体外可强烈激发同种异体T淋巴细胞增殖的树突状细胞群。     

Relative efficacy of human monocytes and dendritic cells as accessory cells for T cell replication

Monocyte-specific monoclonal antibodies (7) were used to compare the efficacy of monocytes and dendritic cells as accessory or stimulator cells for human T cell replication. Both unfractionated and plastic-adherent mononuclear cells were first treated with a cytolytic antimonocyte antibody that kills greater than 95% of monocytes but not dendritic cells. When tested as stimulators of the mixed leukocyte reaction (MLR) and of oxidative mitogenesis (the proliferation of T cells modified with sodium periodate), the monocyte-depleted cells had normal or enhanced stimulatory capacity. Monocyte-depleted mononuclear cells also proliferated normally to soluble antigens (Candida albicans, tetanus toxoid), even under limiting conditions of cell dose, antigen dose, and culture time. Adherent blood mononuclear cells were next separated into monocyte-enriched and -depleted components using fluoresceinated antimonocyte antibody and the cell sorter. The depleted fraction (less than 2% monocytes by esterase staining and by cytology) contained the dendritic cells and exhibited at least 75% of the accessory activity. The monocyte-rich fraction (approximately 97% esterase positive) stimulated the MLR and oxidative mitogenesis weakly, and was comparable in potency to nonadherent cells. Cell-specific antibodies and complement were also used to prepare dendritic cells that were thoroughly depleted of monocytes and lymphocytes. The dendritic cells (70-80% pure) were potent stimulators of the allogeneic MLR, syngeneic MLR, and tetanus toxoid response, being active at stimulator to responder ratios of 1:100 or less. Taken together with previous studies (1, 2), these experiments indicate that the dendritic cell is the major stimulator of T cell replication in man. The contribution of class II products of the major histocompatibility complex (7) was then evaluated with a new monoclonal, 9.3F10. Accessory function was dramatically inhibited if cells bearing class II antigens were killed with 9.3F10 and complement, or if class II molecules were blocked by the addition of 9.3F10 Fab to the culture medium. The expression of 9.3F10 class II products was therefore studied on purified monocytes and dendritic cells. Most if not all cells in both populations reacted with 9.3F10, and each population exhibited approximately 150,000 125I-Fab 9.3F10 binding sites per cell. Since Ia+ dendritic cells are active accessory cells, but Ia+ monocytes are not, class II products are necessary but not sufficient for the stimulation of T cell proliferation in man.     

脐带血树突状细胞的体外诱导及其特征鉴定

目的：探讨在体外无需预先分离CD34^＋造血祖细胞即可有效诱导脐带血单个核细胞而获得脐带血树突状细胞的方法，并对人脐带血树突状细胞的生物学功能进行鉴定。方法：实验于2003—12／2004—10在吉林省肿瘸防治研究所完成。健康孕妇正常胎儿脐带血由吉林省妇幼保健院提供，孕妇签署知情同意书。无菌采集健康孕妇正常胎儿脐带血50mL左右，肝素抗凝。生理盐水稀释后加入甲基纤维素使终浓度为1g／L，室温沉降45min，取上层富含细胞的血浆层，1500r／min离心10min，弃上清，沉淀加两倍体积的生理盐水稀释，以1：1体积叠加于淋巴细胞分层液上，分离出脐带血中的单个核细胞，体外培养24h后去除悬浮细胞，加入粒细胞-巨噬集落刺激因子、白细胞介素4联合刺激脐带血单个核细胞分化为脐带血树突状细胞。在光镜和透射电镜下观察培养的脐带血树突状细胞的形态学特征。碱性磷酸酶-抗碱性磷酸酶桥联法测量脐带血树突状细胞表面分子CD1α、人类白细胞抗原-DR的表达水平。四甲基偶氮唑蓝法测定不同细胞密度的树突状细胞刺激同种T淋巴细胞增殖的能力。结果：①不同诱导时间体外培养的脐带血树突状细胞形态学观察：倒置显微镜下，淋巴细胞经细胞因子诱导后第3d聚集成均匀散布的细胞聚体，部分细胞变形，胞体拉长；第7d可见不明显突起，细胞形态不规则，呈疏松贴壁生长或悬浮生长；14d集落样生长，具有典型的树突状细胞形态。透射电镜下可见树突状细胞呈不规则形，细胞表面粗糙，突起明显；细胞核呈肾形或马蹄形，核浆比增大；胞浆中富含线粒体，较少溶酶体。②细胞因子诱导前后脐血淋巴细胞表型的变化：诱导前脐带血淋巴细胞表面不表达CD1α，诱导后CD1α的表达上升为（22．00&;#177;4．36）％；人类白细胞抗原-DR由诱导前的（3．67&;#177;2．08）％表达上升为（61．67&;#177;7．02）％。③脐带血树突状细胞对T淋巴细胞增殖能力的影响：脐带血树突状细胞具有明显刺激同种T淋巴细胞增殖的功能，且其数量的不同对T淋巴细胞的刺激能力亦不同。脐带血树突状细胞与淋巴细胞比值为1：10对平均刺激指数为3．95，比值为1：100时平均刺激指数为2．16。结论：采取粒细胞-巨噬集落刺激因子、白细胞介素-H4联合刺激可成功诱导出形态典型、纯度较高的脐带血树突状细胞，且体外培养后具有明显的刺激同种T淋巴细胞增殖的能力。     

脐带血树突状细胞的体外诱导及其特征鉴定

目的:探讨在体外无需预先分离CD34 造血祖细胞即可有效诱导脐带血单个核细胞而获得脐带血树突状细胞的方法,并对人脐带血树突状细胞的生物学功能进行鉴定。方法:实验于2003-12/2004-10在吉林省肿瘤防治研究所完成。健康孕妇正常胎儿脐带血由吉林省妇幼保健院提供,孕妇签署知情同意书。无菌采集健康孕妇正常胎儿脐带血50mL左右,肝素抗凝。生理盐水稀释后加入甲基纤维素使终浓度为1g/L,室温沉降45min,取上层富含细胞的血浆层,1500r/min离心10min,弃上清,沉淀加两倍体积的生理盐水稀释,以1∶1体积叠加于淋巴细胞分层液上,分离出脐带血中的单个核细胞,体外培养24h后去除悬浮细胞,加入粒细胞-巨噬集落刺激因子、白细胞介素4联合刺激脐带血单个核细胞分化为脐带血树突状细胞。在光镜和透射电镜下观察培养的脐带血树突状细胞的形态学特征。碱性磷酸酶-抗碱性磷酸酶桥联法测量脐带血树突状细胞表面分子CD1α、人类白细胞抗原-DR的表达水平。四甲基偶氮唑蓝法测定不同细胞密度的树突状细胞刺激同种T淋巴细胞增殖的能力。结果:①不同诱导时间体外培养的脐带血树突状细胞形态学观察:倒置显微镜下,淋巴细胞经细胞因子诱导后第3d聚集成均匀散布的细胞聚体,部分细胞变形,胞体拉长;第7d可见不明显突起,细胞形态不规则,呈疏松贴壁生长或悬浮生长;14d集落样生长,具有典型的树突状细胞形态。透射电镜下可见树突状细胞呈不规则形,细胞表面粗糙,突起明显;细胞核呈肾形或马蹄形,核浆比增大;胞浆中富含线粒体,较少溶酶体。②细胞因子诱导前后脐血淋巴细胞表型的变化:诱导前脐带血淋巴细胞表面不表达CD1α,诱导后CD1α的表达上升为(22.00±4.36)%;人类白细胞抗原-DR由诱导前的(3.67±2.08)%表达上升为(61.67±7.02)%。③脐带血树突状细胞对T淋巴细胞增殖能力的影响:脐带血树突状细胞具有明显刺激同种T淋巴细胞增殖的功能,且其数量的不同对T淋巴细胞的刺激能力亦不同。脐带血树突状细胞与淋巴细胞比值为1∶10时平均刺激指数为3.95,比值为1∶100时平均刺激指数为2.16。结论:采取粒细胞-巨噬集落刺激因子、白细胞介素-4联合刺激可成功诱导出形态典型、纯度较高的脐带血树突状细胞,且体外培养后具有明显的刺激同种T淋巴细胞增殖的能力。     

小鼠髓系树突状细胞的体外扩增和超微结构

背景:树突状细胞可激发初始型T细胞,是目前所知机体功能最强的专职抗原递呈细胞,但对其超微结构的研究鲜见报道.目的:观察小鼠髓系树突状细胞不同发育阶段以及CD40配基化和肿瘤坏死因子α刺激后树突状细胞的超微结构特征.方法:无菌取小鼠骨髓前体细胞,采用粒细胞一巨噬细胞集落刺激因子和白细胞介素4联合方案体外诱导获得未成熟树突状细胞,负载早期凋亡的肿瘤细胞后采用小鼠CD40L转基因CHO细胞和肿瘤坏死因子α刺激48 h,按常规方法制备超薄切片,透射电镜观察树突状细胞的超微结构特征.结果与结论:前体细胞及未成熟树突状细胞内有清晰可见的吞饮泡,未成熟树突状细胞的胞质内可见"C"形和环形溶酶体;凋亡肿瘤细胞负载的树突状细胞可见凋亡小体被吞噬或包裹的现象,小鼠CD40L转基因CHO细胞和肿瘤坏死因子α均可促进树突状细胞成熟,但肿瘤坏死因子α作用后,部分树突状细胞有自噬和凋亡改变.结果提示,小鼠骨髓来源树突状细胞在不同分化发育阶段有其特殊的超微结构表现,肿瘤坏死因子α可介导树突状细胞发生自噬和凋亡.