体外诱导小鼠胚胎干细胞分化为心肌细胞的初步研究

目的：5-氮胞苷作为分化诱导剂，初步探讨其单独或联合全反式维甲酸应用时对小鼠胚胎干细胞（mESC）分化为心肌细胞的影响，旨在建立一种体外诱导mESC分化为心肌细胞的实验方法。方法： 采用 MTT法确定5-氮胞苷的非细胞毒性参考剂量。设计不同条件培养基（5-氮胞苷单独或配伍全反式维甲酸应用）对mESC 进行诱导分化，并通过免疫组化技术及RT-PCR方法等对分化细胞进行鉴定。结果： 5-氮胞苷的非细胞毒性参考剂量为8 μmol/L，能够诱导mESC分化为心肌合胞体（与阴性对照组比较，P<0.01），诱导分化率可达50%。配伍全反式维甲酸持续诱导的结果等同于单独应用全反式维甲酸的作用效果（P>0.05）：即对ESC向心肌细胞的诱导分化没有促进作用。结论：5-氮胞苷能够诱导mESC分化为心肌合胞体，从而得以建立一种体外诱导mESC分化为心肌细胞的方法。     

ω-3多不饱和脂肪酸对树突状细胞生物物理学特性及迁移能力的影响

目的 从生物物理学与免疫学交叉角度,分析二十碳五烯酸(eicosapentaenoic acid, EPA)、二十二碳六烯酸(docosahexaenoic acid, DHA)对鼠源树突状细胞(dendritic cells, DCs)生物物理学特性、细胞骨架及迁移能力的影响,探讨ω-3多不饱和脂肪酸(ω-3 polyunsaturated fatty acids,ω-3 PUFAs)对DCs免疫功能的影响及其潜在作用机制。方法 分离C57BL/6J小鼠骨髓来源单核细胞,经20 ng/mL重组鼠集落刺激因子(recombinant mouse granulocyte macrophage colony stimulating factor, rmGM-CSF)和10 ng/mL重组鼠白介素-4(recombinant mouse interleukin-4, rmIL-4)诱导分化为未成熟树突状细胞(immature dendritic cells, imDCs),第6天加入100 ng/mL脂多糖诱导为成熟树突状细胞(mature dendritic cells, mDCs),进一...     

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。     

全反式维甲酸对TGF-β体外诱导大鼠Foxp3~+Treg分化的作用

目的探索TGF-β、全反式维甲酸及IL-2在大鼠初始T细胞向Foxp3+T细胞分化中的作用。方法分选Lewis大鼠脾脏CD4+CD25-初始T细胞,在抗CD3和CD28抗体激活下,分别用TGF-β、IL-2及全反式维甲酸诱导,通过流式细胞仪检测细胞表面CD25及胞内Foxp3的表达。并提取细胞mRNA通过逆转录PCR检测Treg功能相关基因的表达。结果 TGF-β在体外能诱导大鼠CD4+CD25-初始T细胞分化为CD4+CD25+Foxp3+T细胞,而IL-2或全反式维甲酸的单独作用不能诱导Foxp3表达。IL-2与全反式维甲酸能增强TGF-β诱导Foxp3表达的作用,在TGF-β、IL-2及全反式维甲酸的共同作用下,诱导的Foxp3+T细胞比例提高。TGF-β诱导的Foxp3+T细胞较高表达IL-10、CTLA-4等调节性T细胞功能相关基因。结论 TGF-β在体外能诱导大鼠CD4+CD25-初始T细胞分化为CD4+CD25+Foxp3+T细胞,IL-2与全反式维甲酸能增强这一作用。     

钙载体诱导慢性髓系白血病细胞分化成树突状细胞

目的：探讨用钙离子载体（Calcium ionorphore，CI）A23187诱导慢性髓系白血病（CML）病人来源的白血病细胞分化成树突状细胞（Dendritic cells，DCs）的方法和条件。方法：选择外周血白细胞计数较高的CML病人，采集外周血或骨髓液分离单个核细胞，通过两次贴壁法除去单核细胞和淋巴细胞，然后放入含或不含GM-CSF（200ng／ml）和CI（375ng／ml）的RP-M11640培养液中培养96小时以上，通过流式细胞仪分析细胞表型、倒置显微镜和电镜观察细胞的形态变化来观察CI对白血病细胞诱导成树突状细胞的作用，初步摸索和探讨CI诱导白血病细胞转化成树突状细胞的最佳条件。结果：发现对CML病人外周血或骨髓液分离的白血病细胞加入GM—CSF（200ng／ml）、CI（375ng／ml）和含10％胎牛血清的RPMI1640培养96小时，细胞能获得典型的成熟树突状细胞的形态，CD86、CD80、CD40、CD83和HLA-DR的表达显著提高。结论：CML细胞用GM-CSF和CI联合诱导能获得成熟树突状细胞的典型形态特征和免疫表型。     

黑菇多糖对人白血病细胞系U_937的增殖及分化的实验研究

用黑菇多糖体外处理人单核细胞性白血病细胞分化模型Ｕ９３７细胞，结果表明黑菇多糖明显抑制Ｕ９３７细胞体外增殖（ＩＣ５０２８０μｇ／ｍｌ）并诱导细胞向终末方向分化。经黑菇多糖诱导后的Ｕ９３７细胞，细胞形态发生显著变化，细胞膜标志和生化特征趋向成熟。     

全反式维甲酸诱导小鼠碱性磷酸酶基因启动子区染色体重构

背景：碱性磷酸酶基因是成骨细胞分化和骨形成的重要标志。在C3H10T1/2细胞中,全反式维甲酸可通过核受体上调小鼠碱性磷酸酶的表达,与MAPK通路无关。 目的：从染色体结构调控方面揭示全反式维甲酸上调碱性磷酸酶表达的分子机制。 方法：10^-6 mol/L全反式维甲酸处理C3H10T1/2细胞0,1,6,12 h,DNA酶Ⅰ超敏感实验确定全反式维甲酸调控区域的位置,染色质免疫共沉淀实验检验全反式维甲酸处理细胞后一系列转录相关因子与全反式维甲酸调控区域结合的量效关系以及时相分布。 结果与结论：DNA聚合酶Ⅰ超敏感实验表明,小鼠碱性磷酸酶启动子转录起始位点上游约520 bp附近为潜在的全反式维甲酸调控区域;染色质免疫共沉淀实验表明,全反式维甲酸对小鼠碱性磷酸酶的上调作用是通过一系列转录相关因子的时序性共同作用来实现。表明全反式维甲酸诱导小鼠碱性磷酸酶基因转录的过程中伴随着染色质重构和组蛋白的修饰作用。     

树突状细胞体外成熟及免疫功能的^(18)F-FDG评价CSCD

背景:既往研究表明,不同剂量的电离辐射对树突状细胞的成熟、免疫功能等也可能有一定的影响。目的:分析不同浓度的18F-FDG对体外培养的人外周血单个核细胞源性树突状细胞的成熟及免疫功能的影响。方法:人外周血单个核细胞源性树突状细胞诱导成熟后,分为5组,分别加入含PBS及92.5×104,185×104,370×104,740×104 Bq/mL浓度的18F-FDG培养基,作用24 h后收集细胞,检测树突状细胞的凋亡率、细胞表型(CD1α、CD80、CD83、CD86、HLA-DR)表达、同种混合淋巴细胞反应及细胞培养上清液中的巨噬细胞炎性蛋白1α和单核细胞趋化因子1表达。结果与结论:740×104 Bq/mL浓度的18F-FDG可以引起树突状细胞的凋亡、CD86的表达下调,同种T细胞抗原提呈能力下降,单核细胞趋化因子1的分泌下降;其余浓度的18F-FDG对树突状细胞的成熟及免疫功能无明显影响。结果表明较低浓度的18F-FDG对树突状细胞的凋亡、成熟、抗原提呈、迁移能力等影响较小,可以在选择合适浓度的前提下作为一种树突状细胞细胞的体外标记物。     

人外周血树突状细胞培养和地塞米松对其分化的影响作用

目的分离培养和鉴定人外周血树突状细胞（DC）,以及探讨地塞米松对其分化的影响作用。方法密度梯度离心法分离人外周血单个核细胞,贴壁后加入GM-CSF、IL-4和LPS培养,部分组另加入地塞米松,观察细胞形态学、流式标志和DC与T淋巴细胞共培养后的增殖变化。结果外周血单核细胞诱导培养后具有DC形态学特征,CD83表达上调,CD14表达下调,DC与T淋巴细胞共培养后呈增殖反应。培养液中加入地塞米松后CD83表达下调,CD14表达上调,DC与T淋巴细胞共培养后增殖反应减弱。结论外周血单核细胞经联合细胞因子可诱导为DC;地塞米松可使DC在功能上处于不成熟状态。     

紫草素对人外周血单核细胞来源的树突状细胞表型及功能的影响

目的探讨紫草素对人外周血单核细胞来源的树突状细胞表型及功能的影响。方法从健康人外周血中分离获得单个核细胞,经过夜贴壁后获得单核细胞,体外采用多种细胞因子联合诱导,获得了分化与功能相对成熟的树突状细胞。采用流式细胞仪检测技术观察不同质量浓度紫草素对DC表面分子表达的影响,采用CCK-8法观察紫草素对树突状细胞促淋巴细胞增殖以及ELISA法检测其对树突状细胞分泌细胞因子IL-23的干预作用。结果 TNF-α25 ng/ml刺激树突状细胞,细胞表面分子CD80、CD83表达升高,刺激同种异体淋巴细胞增殖能力增强;100μg/ml紫草素可抑制CD80及CD86的表达,50μg/ml紫草素可抑制CD86的表达;100μg/ml紫草素和50μg/ml紫草素均可抑制树突状细胞促淋巴细胞增殖的能力;100μg/ml紫草素和50μg/ml紫草素均可抑制LPS和INF-γ联合诱导的树突状细胞IL-23的分泌。结论紫草素具有一定的抑制树突状细胞成熟及抑制其分泌IL-23的作用。     

The role of the Fgr tyrosine kinase in the control of the adhesive properties of U937 monoblastoid cells and their derivatives.

In humans, expression of the cellular proto-oncogene c-fgr is normally restricted to mature cells of the myeloid lineage, mantle zone B cells and various myeloid and B-cell lines. Previous studies of the monoblastoid cell line, U937, showed that c-fgr expression increased following differentiation, but its role in monocytes and related cells has not been defined in functional terms. We therefore investigated the role of c-fgr in U937 cells transfected with the c-fgr gene such that its expression could be manipulated independent of differentiation. Induction of the transfected c-fgr gene by cadmium ions did not affect cell proliferation, responses to phorbol 12-myristate 13-acetate (PMA), dihydroxycholecalciferol (DHCC), tumour necrosis factor-alpha (TNF-alpha) or retinoic acid, or phagocytosis of antibody-coated sheep red blood cells. However, there was increased surface expression of CD54 (intracellular adhesion molecule-1; ICAM-1) and CD102 (ICAM-2) and decreased surface expression of CD50 (ICAM-3) compared with cells that had been transfected with plasmid only and treated in the same way. These findings suggest that the product of the c-fgr gene may be important in control of relative adhesive properties of mature monocytic cells.     

1,25-Dihydroxyvitamin D3, but not retinoic acid, induces the differentiation of U937 cells.

We have examined the effects of vitamin D3 metabolites and retinoic acid on the myelomonocyte cell line U937. Inhibition of proliferation, measured by incorporation of 125iodo-deoxyuridine was seen at 72 h with 1,25-(OH)2D3 but not 25(OH)D3 or 24, 25(OH)2D3 metabolites. CD14 molecules, not normally present on U937 cells, were induced on the cell surface. However, Class II major histocompatibility complex (MHC) molecules were not induced and Class I MHC molecules not increased in density as determined by flow cytometry. Retinoic acid inhibited proliferation but failed to induce CD14 molecules. These data suggest that both 1,25(OH)2D3 and retinoic acid act as an antiproliferation signal to U937 cells; only 1,25-(OH)2D3 induces the differentiation towards a more mature phenotype.     

A 93-kDa glycoprotein expressed on human cultured monocytes and dendritic reticulum cells defined by an anti-K562 monoclonal antibody

An IgG2a monoclonal antibody, referred to as 12B1 and raised against the K562 cell line, reacted with adherent monocytes maintained in culture for several days but not with bone marrow or peripheral blood cells including freshly isolated monocytes. Among human leukemic cell lines, 12B1 reacted essentially with the promyelocytic HL60 cell line. 12-O-Tetradecanoylphorbol 13-acetate treatment, but no other differentiation inducer, strongly enhanced its reactivity on K562, HL60 and the histiocytic U937 cell line. Immunoperoxidase staining of sections of normal human tissues showed that 12B1 specifically recognized dendritic reticulum cells in germinal centers of lymph nodes, spleen and tonsils. The 12B1-detected antigen is a highly glycosylated polypeptide of an apparent molecular mass of 93-86 kDa. The 12B1 antigen appears to be a new glycoprotein marker shared by adherent monocytes and dendritic reticulum cells. The association of the 12B1 epitope with cells which present antigen and/or exert accessory function suggests that this molecule could play a role in these activities.     

人CD83基因转染细胞株的构建及其功能初步研究

目的构建人CD83全长编码基因转染293细胞,并探讨其对单核细胞的作用。方法 RT-PCR方法从成熟人树突状细胞(DC)扩增出CD83 cDNA,插入pIRES2-EGFP载体,脂质体转染293细胞,筛选出稳定表达目的基因细胞株;从人外周血单个核细胞(PBMC)中磁珠分选单核细胞,LPS刺激的同时加入293/CD83细胞或293/mock,24 h后检测细胞活化状态以及培养上清中TNF-α水平。结果经流式细胞术反复检测绿色荧光蛋白(GFP)报告基因及CD83表达水平,筛选获得获得一株稳定表达膜型CD83分子的基因转染细胞株293/CD83;体外实验显示293/CD83可促进LPS刺激的单核细胞活化水平和TNF-α分泌量。结论成功构建表达人CD83分子的293细胞株,293/CD83对LPS刺激的单核细胞有协同刺激作用。     

Retinoic acid‐producing,ex‐vivo‐generated human tolerogenic dendritic cells induce the proliferation of immunosuppressive B lymphocytes

While much is known about tolerogenic dendritic cell effects on forkhead box protein 3 (FoxP3)⁺ regulatory T cells, virtually nothing is known about their effects on another arm of immunoregulation that is mediated by a subpopulation of immunosuppressive B cells. These cells suppress rheumatoid arthritis, lupus and inflammatory bowel disease in mice, and functional defects have been reported in human lupus. We show that co‐stimulation‐impaired tolerogenic dendritic cells that prevent and reverse type 1 diabetes mellitus induce the proliferation of human immunosuppressive B cells in vitro. We also show that the suppressive properties of these B cells concentrate inside the CD19⁺CD24⁺ B cell population and more specifically inside the CD19⁺CD24⁺CD38⁺ regulatory B cell population. We discovered that B cell conversion into suppressive cells in vitro is partially dependent on dendritic cell production of retinoic acid and also that CD19⁺CD24⁺CD38⁺ B regulatory cells express retinoic acid receptors. Taken together, our data suggest a model whereby part of the immunosuppressive properties of human tolerogenic dendritic cells could be mediated by retinoic acid which, in addition to its known role in favouring T cell differentiation to FoxP3⁺ regulatory T cells, acts to convert B cells into immunosuppressive cells.     

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。 关键词：明胶;单核细胞;树突状细胞;人外周血;单个核细胞 doi:10.3969/j.issn.1673-8225.2012.10.037     

Airway epithelial IL-15 transforms monocytes into dendritic cells

IL-15 has recently been shown to induce the differentiation of functional dendritic cells (DCs) from human peripheral blood monocytes. Since DCs lay in close proximity to epithelial cells in the airway mucosa, we investigated whether airway epithelial cells release IL-15 in response to inflammatory stimuli and thereby induce differentiation and maturation of DCs. Alveolar (A549) and bronchial (BEAS-2B) epithelial cells produced IL-15 spontaneously and in a time- and dose-dependent manner after stimulation with IL-1beta, IFN-gamma, or TNF-alpha. Airway epithelial cell supernatants induced an increase of IL-15Ralpha gene expression in ex vivo monocytes, and stimulated DCs enhanced their IL-15Ralpha gene expression up to 300-fold. Airway epithelial cell-conditioned media induced the differentiation of ex vivo monocytes into partially mature DCs (HLA-DR+, DC-SIGN+, CD14+, CD80-, CD83+, CD86+, CCR3+, CCR6(+), CCR7-). Based on their phenotypic (CD123+, BDCA2+, BDCA4+, BDCA1(-), CD1a-) and functional properties (limited maturation upon stimulation with LPS and limited capacity to induce T cell proliferation), these DCs resembled plasmacytoid DCs. The effects of airway epithelial cell supernatants were largely blocked by a neutralizing monoclonal antibody to IL-15. Thus, our results demonstrate that airway epithelial cell-conditioned media have the capacity to differentiate monocytes into functional DCs, a process substantially mediated by epithelial-derived IL-15.     

用GM-CSF和 IL-4在体外诱导高纯度CD14+树突状细胞

本研究改进传统的树突状细胞（DC）诱导方法，用GM－CSF（150ng/ml）和IL－4（80U／ml），在体外经7d从健康人外周血中诱导出了大量高纯度DC，其高表达HLA－I、HLA－II类分子，共刺激分子和黏附分子，同时还高表达其前体单核细胞的特异性标志CD14分子，显示出成熟 DC的特征。这些CD14^ DC能强烈诱导同种异体淋巴细胞的增殖，其内吞能力在第3天达最高，之后明显下降。此结果丰富了DC的类型，并为CD14^ DC的深入研究和应用奠定了基础。     

Circulating human dendritic cells differentially express high levels of a 55-kd actin-bundling protein.

This study was initiated to examine the differential expression of an evolutionary conserved human 55-kd actin-bundling (p55) protein that is induced in B lymphocytes by Epstein-Barr virus infection. Our study demonstrates that p55 is specifically expressed at constitutively high levels in human peripheral blood dendritic cells and lymph node (interdigitating) dendritic cells. Blood dendritic cells constitute a minority (< 2%) of all blood leukocytes but are a distinct population of potent antigen-presenting cells. Immunofluorescence microscopy with a monoclonal antibody specific for p55 showed that 87% of peripheral blood dendritic cells stained brightly in the cytoplasm and in the veiled cytoplasmic extensions. In contrast, monocytes, granulocytes, T cells, and B lymphocytes showed no expression of the p55 protein. Western blot analysis confirmed that only the dendritic cell component of peripheral blood expressed high levels of p55. Staining of human lymph node sections demonstrated selective expression of the p55 antigen by dendritic cells in the T-cell-dependent areas but not in the B cell follicles. p55 is likely to be involved in the organization of a specialized microfilament cytoskeleton in the dendritic cells, and the anti-p55 antibody should be useful for further characterization of this important population of antigen-presenting cells in clinical transplantation, HIV-1 pathogenesis, and autoimmune diseases.