体外诱导成熟树突状细胞的研究

目的：探讨在体外从干细胞中诱导出成熟DC的适宜环境。方法：分别将人胎肝、骨髓和脾细胞以及小鼠骨髓和脾细胞在体外用GM－CSF和TNF－α诱导,观察了第3、5、7天DC的收获情况。进一步用S－P免疫细胞化学染色检测了mBmDC和fLDC有关分子表达。结果：在体外通过GM－CSF和TNF－α的作用,小鼠骨髓、人胎肝、骨髓细胞呈典型的毛刺状胞浆突起,第5天的收获率分别为：39.5％、67.2％、12.9％,而基本不能从脾细胞中诱导出成熟DC,获得的DC能与MAbCD80、MAbCD40呈强阳性反应。结论：在GM－CSF和TNF－α的共同作用下,能在体外从人胎肝、 骨髓和小鼠骨髓干细胞中获得成熟DC,其DC能表达高高水平的B7－1和CD40分子,这为大量获得DC提供了一种简便可行的手段,为研究DC的生物学特征及其抗原提呈功能提供了丰富的材料,为开展以DC为基础的各种免疫治疗研究奠了良好的基础。     

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。     

成人外周血来源树突状细胞体外诱导培养及成熟调控

人体内树突状细胞具有成熟和未知成熟两种形态。前者是早期免疫应答的强有力的抗原递呈细胞。后者具有截然不同的生物学特性，且能诱导免疫耐受。本研究从成人外周血中分离前体细胞，利用单核细胞条件性培养液，培养出比较均一的未成熟及成熟阶段的树突状细胞。第一阶段：从外周血分离出能粘附塑料的单核细胞，在GM－CSF＋IL-4存在条件下培养6－7d；第二阶段，加入单核细胞条件性培养液促进树突状细胞分化成熟，仅经第一阶段培养 的细胞主要为未成熟树突状细胞，仍然表达单核细胞的表达标志CD14，具有活跃的内,骐 MHC－II分子主要分布在胞内的MIIC器官；经第二阶段培养的树突状细胞则具有成熟树突状细胞的全部特征：典型的树突状形态，悬浮生长，MHC－II分子主要分布在包膜表面，．表达树突状细胞的特异性标志CD83，刺激同种T淋巴细胞增殖的能力强，并在脱离特定细胞因子环境3d后仍能保持该性状不变。由于该途径培养成熟、未成熟树突状细胞完全受外部因素调控，可能是获得均一的成熟和未成熟树突状细胞供科研和临床应用的较好途径。     

小鼠胸腺树突状细胞的分离和纯化方法的改进

目的　探讨一种简便易行的小鼠胸腺树突状细胞（ＴＤＣ）的分离纯化方法。　方法　密度梯度离心结合小鼠ＣＤ５ 单克隆抗体（ｍ Ａｂ）淘洗法（ｐａｎｎｉｎｇ）。　结果　细胞浓度在１．５×１０７ ／ｍ ｌ时进行Ｐｅｒｃｏｌｌ密度梯度离心可使分离纯度高、细胞丢失少；依次使用两种分离液分离的效果优于同一种分离液的反复使用；小鼠ＣＤ５单抗浓度为２５ｍ ｇ／Ｌ时，淘洗纯化效果较好，ＴＤＣ纯度达７５％ ～９０％ 。　结论　密度梯度离心与ＣＤ５单抗淘洗纯化的方法切实可行。     

体外培养树突状细胞成熟诱导物的研究进展

树突状细胞(DC)是体内功能最强的抗原提呈细胞(APC),参与多种自身免疫疾病、肿瘤、免疫缺陷病及某些炎症反应的病理过程.以成熟DC为基础制备的DC疫苗具有明显的诱发抗肿瘤免疫应答的能力,其显著的抗肿瘤效应已经得到广泛认可.目前体外培养DC的方法尚没有统一的标准,致使培养得到的DC在均一度和功能上有很大的差别,很大程度上限制了DC的基础研究和DC疫苗的临床应用工作的开展.以下综述了体外扩增DC所用成熟诱导物的特点,为相关研究者体外扩增DC提供了参考.     

脐血树突状细胞的诱导和体外抗肿瘤研究

目的：用细胞因子诱导脐血干细胞分化为树突状细胞(dendritic cells，DC)，并观察脐血来源DC疫苗在体外对人肝癌细胞的杀伤活性。方法：用CD34^ 细胞分选试剂盒和Mini MACS从脐血单个核细胞中分离CD34^ 干细胞。重组人粒细胞-巨噬细胞集落刺激因子100μg／ml、重组人肿瘤坏死因子-α50U／ml诱导CD34^ 干细胞向DC分化，     

未成熟树突状细胞快速分离与体外诱导培养及其鉴定研究

目的:以人外周血来源的单核细胞为前体细胞,建立体外快速分离和诱导培养未成熟树突状细胞(Immature dendritic cell,iDC)的方法。方法:采用Ficoll 密度梯度离心方法和MACS 磁珠分选系统,收集高纯度CD14+ 单核细胞;用rhGM-CSF、rhIL-4 联合诱导培养CD14+单核细胞,第4 天获得未成熟树突状细胞(iDC),应用流式细胞术检测细胞表面标记(CCR5)和抗原吞噬能力,普通光学显微镜、扫描电镜和透射电镜观察细胞表面和内部结构特征。结果:CD14 免疫磁珠技术获得CD14+单核细胞纯度达94%以上,诱导分化第4 天的iDC 具有CCR5 特征性标志和吞噬能力,普通光学显微镜及电镜观察到iDC 出现树突状细胞典型特征。结论:体外快速诱导培养是获得大量具有典型特征的iDC 的有效方法,并可应用于进一步实验研究。     

人外周血分离树突状细胞关体分离培养及其分化成熟过程中生物特征 …

用从人外周血分离。纯化，扩增树突状细胞的方法，对树突状细胞成熟过程中的功能化作初步探讨。以酵母菌，ＨＲＰ吞噬实验测试树突状细胞的内吞能力；以同种混合淋巴细胞反应检测细胞的免疫刺激能力。     

体外诱导扩增大鼠树突状细胞及其特性鉴定

目的: 体外诱导和扩增大鼠骨髓树突状细胞 (DC)的方法, 并进行生物学特性鉴定。方法: 将大鼠骨髓细胞培养 48h后, 去除悬浮细胞, 加入IL- 4和GM- CSF培养 2wk。在光镜和透射电镜下观察培养的DC的形态学特征。应用流式细胞仪检测DC表面分子MHC-Ⅱ类分子、B7 -1、B7- 2的表达水平。将DC与同种异体T细胞混合培养, 采用MTT比色法测定不同细胞密度的DC刺激同种T细胞增殖的能力。结果: 培养的DC胞浆突起大而长, 呈树突状, 具有DC的典型形态。培养 2wk的DC表面MHC -Ⅱ类分子表达的阳性率为 74. 2%, B7- 1、B7 -2分别为 81%、76%。培养的DC具有明显刺激同种异体T细胞增殖的能力。结论: 将大鼠骨髓细胞经贴壁去除悬浮的细胞, 加入IL -4和GM -CSF培养, 可获得足量、较高纯度的DC, 为研究DC的功能奠定了基础。     

树突状细胞的分离扩增与培养技术研究进展

树突状细胞 (Dendriticcells,DC)因其成熟时伸出许多树突样或伪足样突起而得名。目前一致认为 ,具有典型树突状形状 ,膜表面高表达MHCⅡ类分子 ,能移行至淋巴器官和刺激初始型T细胞增殖活化 ,并具有一些相对特异性表面标志的一类细胞 ,方能称之为DC。有髓系来源DC与淋巴系干细胞来源DC之分 ,大多数DC来源于骨髓 ,DC前体细胞由骨髓进入外周血 ,再分布到全身各组织。DC是目前已知的功能最强的抗原呈递细胞 ,参与抗原的识别、加工处理和提呈 ,是机体免疫反应的始动者。其最大的特点是能够显著刺激处女型或初始型T细胞增殖 ,并能过其表面大量的MHC与抗原联合 ,以膜结合的方式递呈抗原 ,DC与T细胞聚合成簇 ,为抗原递呈和T细胞增殖提供稳定的微环境 ,从而参与多种自身免疫疾病、肿瘤、免疫缺陷病及某些炎症反应的病理过程 ,在免疫应答的诱导中具有独特地位。从直接分离提取到添加细胞因子诱导扩增 ,DC的研究获得很大进展 ,成为当今国内外免疫学及其相关学科的研究热点     

丁硫氨酸硫酸亚胺早期处理抑制单核细胞向树突状细胞的分化

 目的 研究细胞氧化-还原态对树突状细胞（DC）分化和功能的影响。 方法 细胞氧化-还原态的调控应用特异性谷胱甘肽（GSH）合成抑制剂丁硫氨酸硫酸亚胺（BSO）。自健康人外周血单个核细胞（PBMC）分离单核细胞，在含重组人粒细胞-巨噬细胞集落刺激因子和 IL-4的RPMI 1640 培养液中诱导分化 DC。将单核细胞分为 BSO 早期（培养第 1 天）用药组、BSO 晚期（培养第 5 天）用药组和不予 BSO 处理的对照组。BSO 用药剂量为终浓度 1 mmol/L，换液时补加相应剂量的 BSO。培养第 5 天，分别给予加入脂多糖（LPS）100 ng/ml（LPS刺激组）或重组人干扰素 （rhIFN- ）20 U/ml（IFN- 刺激组）或不予刺激（无刺激组）的处理；培养第 7 天收集各组悬浮细胞进行表型分析和功能检测，并在倒置显微镜下观察 BSO 早期用药组和对照组贴壁细胞。表型分析采用特异性荧光标记单抗及流式细胞仪。功能检测采用混合淋巴细胞反应试验，将经丝裂霉素C 处理的 DC（刺激细胞）与异基因非贴壁 PBMC（反应细胞）混合（刺激细胞:反应细胞分别为 1:25、1:50、1:100）培养 5 d 后，用3H-胸腺嘧啶核苷法检测淋巴细胞增殖情况。 结果 培养第 7 天，镜下观察显示 BSO早期用药组贴壁细胞明显多于对照组；流式细胞仪检测显示 BSO 早期用药组 CD86 和CD1a表达明显低于对照组，CD86 平均荧光强度（MFI）分别为311.3 ± 97.3、552.0 ± 97.9（P = 0.01），CD1a 分别为52.2 ± 22.2、121.2 ± 4.2（P = 0.03）；BSO早期用药组及对照组3H-胸腺嘧啶核苷掺入量（cpm）在刺激细胞:反应细胞＝1:25时分别为 1587 ± 1458 和9336 ± 1333（P=0.015）；加入rhIFN- 20 U/ml 刺激后二组 CD86 MFI 分别为 495.9 ± 143.9 和 800.4 ± 27.7（P = 0.06），而加入 LPS 100 ng/ml 刺激后二组CD86 MFI 分别为 1736.7 ± 375.7 和 1960.8 ± 494.5（P > 0.05）。表明单核细胞分化早期加入 BSO 对 DC 的分化和功能有抑制作用，并对 IFN- 诱导DC成熟有抑制作用。BSO 晚期用药组 DC 抗原提呈功能分子的表达及与对照组比较均无明显差异，对 LPS 或 rhIFN- 刺激的 DC成熟也无明显影响。 结论 BSO 早期持续处理能抑制单核细胞向 DC 细胞的分化，细胞内氧化-还原态水平可能是影响 DC 分化成熟的关键因子。     

Aire deficient dendritic cells promote the T follicular helper cells differentiation

Autoimmune regulator (Aire), primarily expressed in medullary thymic epithelial cells (mTECs), maintains central immune tolerance through the clearance of self-reactive T cells. Aire can also be expressed in dendritic cells (DCs), and DCs can mediate T follicular helper (T_FH) cell differentiation and self-reactive B cell activation through inducible costimulator molecule ligand (ICOSL) and interleukin 6 (IL-6), which can cause autoimmune diseases. To confirm whether Aire in DCs affects T_FH cell differentiation and to determine the role of Aire in the maintenance of peripheral immune tolerance, this study observed the effects of Aire deficiency on T_FH cells using Aire knockout mice. The results showed that Aire deficiency caused increased number of T_FH cells, both in vivo and in vitro. Further studies showed that Aire deficiency promoted T_FH differentiation through the upregulation of ICOSL and IL-6 in DCs. Thus Aire could suppress the expression of ICOSL and IL-6 to inhibit T_FH cell differentiation.     

Notch signaling in differentiation and function of dendritic cells

Hematopoietic stem cells give rise to multiple lineages of cells. This process is governed by a tightly controlled signaling network regulated by cytokines and a direct cell-cell contact. Notch signaling represents one of the major pathways activated during direct interaction between hematopoietic progenitor cells and bone marrow stroma. A critical role of Notch signaling in differentiation of T- and B-lymphocytes has now been established. Until recently, the role of Notch signaling in the development of myeloid cells and particular dendritic cells remained unclear. In this review, we discuss recent exciting findings that shed light on the critical role of Notch in differentiation and the function of dendritic cells and its impact on immune responses.     

脐血CD34+细胞体外定向诱导分化为T淋巴细胞的实验研究

目的：建立利用人造血干／祖细胞体外定向诱导分化为T淋巴细胞的方法，为研究T细胞生物学特性及细胞免疫提供技术平台。方法：MACS方法分离人脐带CD34^ 细胞接种到人胎儿胸腺基质单层细胞上，IMDM液体培养基含20％人AB血清并加入FL、IL-12、IL-7和IL-2细胞因子组合，于培养7、14、21、28、35、42天取非贴壁细胞利用流式细胞仪对细胞表型进行检测，并进行细胞形态学分析。结果：2周后，CD4^ CD8^ 非成熟T淋巴细胞占细胞总数的0．3％-13．3％，4-5周CD4^ CD8^ T淋巴细胞达到高峰占16．6％-26．5％，且CD3^ CD4^ CD8^ 和CD3^ CD4^-CD8^ T淋巴细胞逐渐增多，6周后达26．5％～64．9％和11．6％-38．9％。培养成熟的T淋巴细胞经PHA IL-2刺激后瑞氏染色鉴定可见大原始淋巴细胞存在。结论：利用人脐血CD34^ 在体外人胎儿胸腺基质单层细胞上加FL、IL-12、IL-7和IL-2细胞因子组合条件下，可诱导分化出T淋巴细胞，并且培养的T细胞对有丝分裂素刺激有增殖反应。     

Selenium accelerates chicken dendritic cells differentiation and affects selenoproteins expression

Selenium (Se) promotes immune cell differentiation and improves immune response. Antigen-presenting cells such as dendritic cells (DCs) play an important role in immune system, however, the impact of Se on DCs is still unclear. In this study, we successfully induced and cultured chicken DCs from peripheral blood mononuclear cells by incubating mononuclear cells with 50 ng/mL recombinant chicken granulocyte-macrophage colony stimulating factor and 10 ng/mL recombinant chicken interleukin-4 for total 9 days. In + Se group, we added 10⁻⁷ mol/L sodium selenite from the first day of cell culture. The results showed that Se supplementation expedited and increased the expression of cell surface markers including CD11c, CD40, CD86, and MHC II. Principal component analysis showed that the expression of selenoproteins SelW, SelK, Dio3, GPX1, GPX2, SelN, SelS, SelH in chicken DCs was highly correlated, and SelW had highest correlation with the cell surface markers MHC II and CD11c. In conclusion, Se accelerates the differentiation and maturation of chicken DCs. Se regulates the differentiation and maturation of chicken DCs by selenoproteins. Selenoproteins has closely correlated to surface markers of chicken DCs.     

Impaired dendritic cell differentiation of CD16‐positive monocytes in tuberculosis: Role of p38 MAPK

Tuberculosis (TB) is one of the world's most pernicious diseases mainly due to immune evasion strategies displayed by its causative agent Mycobacterium tuberculosis (Mtb). Blood monocytes (Mos) represent an important source of DCs during chronic infections; consequently, the alteration of their differentiation constitutes an escape mechanism leading to mycobacterial persistence. We evaluated whether the CD16⁺/CD16^? Mo ratio could be associated with the impaired Mo differentiation into DCs found in TB patients. The phenotype and ability to stimulate Mtb‐specific memory clones DCs from isolated Mo subsets were assessed. We found that CD16^? Mos differentiated into CD1a⁺DC‐SIGN^high cells achieving an efficient recall response, while CD16⁺ Mos differentiated into a CD1a^?DC‐SIGN^low population characterized by a poor mycobacterial Ag‐presenting capacity. The high and sustained phosphorylated p38 expression observed in CD16⁺ Mos was involved in the altered DC profile given that its blockage restored DC phenotype and its activation impaired CD16^? Mo differentiation. Furthermore, depletion of CD16⁺ Mos indeed improved the differentiation of Mos from TB patients toward CD1a⁺DC‐SIGN^high DCs. Therefore, Mos from TB patients are less prone to differentiate into DCs due to their increased proportion of CD16⁺ Mos, suggesting that during Mtb infection Mo subsets may have different fates after entering the lungs.     

间充质干细胞调节树突状细胞相关功能的研究进展

树突状细胞(DCs)作为体内最强大的抗原提呈细胞,是适应性免疫应答的始动者,在较多疾病的发生发展过程中起着重要作用.间充质干细胞(MSCs)是具有多向分化潜能的成体干细胞,由于其强大的自我修复、抑制炎症以及免疫调节作用已成为研究的热点.大量的研究表明MSCs能通过影响DCs的成熟及功能,达到缓解疾病的目的.通过对MSCs调节DCs功能调节的深入研究有望取得疾病治疗的新进展.     

Adipose tissue-derived mesenchymal stem cells are more potent suppressors of dendritic cells differentiation compared to bone marrow-derived mesenchymal stem cells

Both mesenchymal stem cells (MSCs) and dendritic cells (DCs) are engaged in the regulation of the immune response parallel to their numerous functions.The main objective of this study was to compare the effects of mesenchymal stem cells isolated from human adipose tissue or human bone marrow on the expression of specific cell surface markers as well as the secretion of some cytokines by monocyte-derived dendritic cells. The set of methods used includes cell cultures, magnetic beads isolation of cells, flow cytometry, ELISA and proteome profiler kit assays. The results obtained show that MSCs isolated from human adipose tissue are more potent immunomodulators of differentiation of human DCs in comparison to the bone marrow-derived MSCs. In both cases the percentages of CD14+ cells were increased in co-cultures of MSCs and DCs and at the same time down-regulated the expression of CD80, CD86 and CD83 as in all experiments the effect of adipose tissue MSCs was stronger. Similarly, the secretion of IL-10 by dendritic cells was up-regulated in co-cultures of MSCs and dendritic cells and the effect was stronger when adipose tissue-derived MSCs were used.Taken together all results presented reveal the higher potential of the adipose tissue-derived MSCs to inhibit the differentiation and expression of functionally important co-stimulatory molecules on the surface of monocyte-derived dendritic cells than the bone marrow-derived MSCs.