树突状细胞:树突状细胞的分布

1.树突状细胞的分布

它们通常少量分布于与外界接触的皮肤(黏膜)部位，主要为皮肤(在皮肤上的，称为Langerhans细胞[注：和鼻腔、肺、胃与肠的内层。血液中也可发现他们的未成熟型式。他们被活化时，会移至淋巴组织中与T细胞与B细胞互相作用，以刺激与控制适当的免疫反应。在一段成长过程中他们长出树枝状的突起，因此本文译为树突状的细胞。这些并不与神经元有特殊的关联，虽然其也有相似的部位。未成熟的树突状细胞也叫做隐匿性细胞，它们具有大量的、细胞质的菌幕。

2.树突状细胞的功能

表达低水平的共刺激因子和粘附因子，体外激发同种混合淋巴细胞增殖反应的能力较低，但未成熟DC具有极强的抗原吞噬能力，在摄取抗原(包括体外加工)或受到某些因素刺激时即分化为成熟DC，而成熟的DC表达高水平的共刺激因子和粘附因子。由接触抗原的外周组织迁移进入次级淋巴器官，与T细胞接触并激发免疫应答。能够诱导特异性的细胞毒性T淋巴细胞(cytotoxic T lymphocyte，应用肿瘤相关抗原或抗原多肽体外冲击致敏DC，可诱发特异性CTL的抗肿瘤免疫反应。大部分实体瘤内浸润的DC数量多则患者预后好。有效的抗肿瘤免疫反应的核心是产生以CD8+ T细胞为主体的细胞免疫应答，这也是DC作为免疫治疗手段的基础。DC抗肿瘤的机制如下：MHC分子与其捕获加工的肿瘤抗原结合，并递呈给T细胞，从而启动MHC-I类限制性CTL反应和MHC-Ⅱ类限制性的CD4+ Thl反应。DC还通过其高表达的共刺激分子(CD80/B7-1、CD86/B7-2、CD40等)提供T细胞活化所必须的第二信号，②DC与T细胞结合可大量分泌IL-12、IL-18激活T细胞增殖，诱导CTL生成，激活穿孔素P颗粒酶B和FasL/Fas介导的途径增强NK细胞毒作用；CCK)专一趋化初始型T细胞促进T细胞聚集，增强了T细胞的激发。保持效应T细胞在肿瘤部位长期存在。

3.dc树突状细胞上清分泌哪些细胞因子

Analysis of T Cell Proliferating and Polarizing Potential of Murine Dendritic Cells in Allogeneic-mixed Leukocyte Reaction建立可同时研究T淋巴细胞增殖和树突细胞极化的共培养体系免疫学 >树突细胞哺乳纲 >脾脏 >基于细胞的分析方法作者:https://doi.org/10.21769/BioProtoc.1750[Abstract] Dendritic cells (DCs) play a critical role in mounting the T cell response against different infectious agents. Nature and intensity of the induced T cell responses are defined by activation status of DCs. It is generally accepted that IL-12,IL-4/these approaches provide indirect information about T cell activating potential of DCs. Analysis of T cell responses in a co-culture system is a more direct approach to examine T cell proliferating and polarizing efficacy of DCs.A protocol to analyze the T cell proliferating and polarizing potential of DCs in an allogeneic mixed leukocyte reaction (allo-MLR) is described here.Materials and ReagentsRPMI-1640 medium (HiMedia Laboratories,TS1006 )Heat-inactivated fetal bovine serum (Biological industries,catalog number:315-03 )Untouched CD4+ anrespectively)Fluorochrome-conjugated FITC anti-mouse CD3,catalog number:respectively)Concanavalin A (Sigma-Aldrich,catalog number:T8154 )RPMI-10 (see Recipes)EquipmentHaemocytometerHumidified CO2 incubatorLaminar air flow bio-safety cabinetCentrifugeGamma-irradiatorMicroscopeFlow-cytometerProcedureHarvest mouse bone marrow-derived dendritic cells (BMDCs) from plates by gentle pipetting,add 4 x 106 bone-marrow cells per well of 6-well plate in RPMI-10 medium supplemented with 20 ng/ml GM-CSF. Remove culture medium along with non-adherent cells on day 3 and day 5,these wells can be filled with autoclaved distillg/ml,CD8 antibodies by flow cytometry. Note:lymphocytes can be prepared from Balb/c mice.Irradiate DCs with gamma-rays in a gamma-irradiation chamber (irradiation dose,which could otherwise give false results.Adjust concentration of lymphocytes to 1.0 x 106 cells/g/ml).Keep plates at 37 °C in a humidified CO2 incubator. After 72 h,transfer plates to -20 °C. Plates can be thawed immediately or next day.Harvest the cells onto a filter paper and wash them using an automated cell harvester. Measure the 3H-thymidine levels on filter paper using a beta scintillation counter.Collect culture supernatants from plate set up withTH17 signature cytokines by ELISA.RecipesRPMI-10RPMI-1640 base medium supplemented with 10% heat-inactivated FBS and 1% antibiotic-antimycotic solutionReferencesKumar,John,V.,S.,Das,and Th1/C.,James,e1750. DOI:BioProtoc.1750;Full Text

4.如何做树突状细胞和t细胞的共培养

Analysis of T Cell Proliferating and Polarizing Potential of Murine Dendritic Cells in Allogeneic-mixed Leukocyte Reaction建立可同时研究T淋巴细胞增殖和树突细胞极化的共培养体系免疫学 > 免疫细胞功能 > 树突细胞哺乳纲 > 鼠科 > 脾脏 > 基于细胞的分析方法作者: Pawan Kumar and Sangeeta Bhaskar Vol 6, Iss 5, 2016/3/5, 1149 views, 0 Q&ADOI: https://doi.org/10.21769/BioProtoc.1750[Abstract] Dendritic cells (DCs) play a critical role in mounting the T cell response against different infectious agents. Nature and intensity of the induced T cell responses are defined by activation status of DCs. It is generally accepted that IL-12, IL-4/IL-5 and IL-23 producing DCs induce TH1, TH2 and TH17 type of immune responses, respectively (Kumar et al., 2015). Besides cytokines, levels of co-stimulatory molecules on DCs also influence the response of T cells.The activation status of DCs can be determined by examining DC culture supernatants for different cytokines and by analyzing expression of co-stimulatory molecules on these cells. However, these approaches provide indirect information about T cell activating potential of DCs. Analysis of T cell responses in a co-culture system is a more direct approach to examine T cell proliferating and polarizing efficacy of DCs.A protocol to analyze the T cell proliferating and polarizing potential of DCs in an allogeneic mixed leukocyte reaction (allo-MLR) is described here.Materials and ReagentsRPMI-1640 medium (HiMedia Laboratories, catalog number: AT028 )Dulbecco’s Phosphate Buffered Saline (HiMedia Laboratories, catalog number: TS1006 )Heat-inactivated fetal bovine serum (Biological industries, catalog number: 04-121-1A ) Antibiotic-antimycotic (penicillin-streptomycin) solution, 100x (HiMedia Laboratories, catalog number: A002A )Round bottom, 96-well cell culture plates (Corning, catalog number: 3799 ) Dendritic cells (derived by culturing mouse bone marrow cells in the presence of recombinant GM-CSF) (PeproTech, catalog number: 315-03 )Untouched CD4+ and CD8+ T cells from allogeneic mouse strain (isolated from spleen of Balb/c mice using CD4 T cell enrichment kit and CD8 T cell enrichment kit (BD, catalog number: 558131 and 558471 , respectively)Fluorochrome-conjugated FITC anti-mouse CD3, PE anti-mouse CD4 and PE anti-mouse CD8 antibodies (BD Pharmingen, catalog number: 555274 , 553730 and553032 , respectively)Concanavalin A (Sigma-Aldrich, catalog number: C5275 ) 3H-thymidine (BARC)Trypan Blue (Sigma-Aldrich, catalog number: T8154 )RPMI-10 (see Recipes)EquipmentHaemocytometerHumidified CO2 incubatorLaminar air flow bio-safety cabinetCentrifugeGamma-irradiatorMicroscopeFlow-cytometerProcedureHarvest mouse bone marrow-derived dendritic cells (BMDCs) from plates by gentle pipetting, give a wash with PBS and prepare the suspensions of 1.0 x 105, 2.0 x 105 and 4.0 x 105 cells per ml in RPMI-10 medium. (BMDCs are derived by culturing mouse bone marrow cells in the presence of GM-CSF. Briefly, add 4 x 106 bone-marrow cells per well of 6-well plate in RPMI-10 medium supplemented with 20 ng/ml GM-CSF. Remove culture medium along with non-adherent cells on day 3 and day 5, and fresh 4.0 ml GM-CSF-supplemented medium to each well. Harvest immature DCs on day 7 by gently pipetting. After giving a wash in RPMI-10 medium, cells can be used for subsequent experiments. Purity of DCs derived following this protocol is ~85%. These cells can be used directly in allo-MLR or can be further purified.)To analyze the ability of DCs to induce T-cell proliferation, add 50 µl of DC suspensions (equivalent to 0.5 x 104, 1.0 x 104 and 2.0 x 104 DCs) per well in a round bottom plate in triplicates.(It is necessary to plate the increasing number of DCs to achieve an increasing ratio of stimulator cells to responder cells. It is advised to not add DCs into outer wells of the plate because culture media tend to evaporate from these wells at higher rates. Instead, these wells can be filled with autoclaved distilled water).To analyze the ability of DCs to induce T-cell polarization, similarly add 50 µl of 2.0 x 105 cells/ml DC suspension (= 1.0 x 104 cells) per well in a round bottom plate in triplicates.Add the desired stimuli such as LPS or heat-killed mycobacteria to plated DCs and adjust final volume of total contents per well to 100 µl. [Dilute stock solution of LPS or mycobacterial suspension to required concentration in RPMI-10 medium. LPS could be used at a concentration of 0.1 to 1.0 µg/ml, whereas heat-killed bacteria (prepared by autoclaving) can be used preferably at a multiplicity of infection (MOI) of 5 to 10].Keep plates in a humidified CO2 incubator for 24 h.Next day, isolate CD4+ and CD8+ T lymphocytes from the spleen of naïve allogeneic mice using a negative selection kit as suggested by manufacturer. Determine purity of lymphocytes using anti-mouse CD3/CD4 and CD3/CD8 antibodies by flow cytometry. Note: If DCs are derived from C57BL/6 mice, lymphocytes can be prepared from Balb/c mice.Irradiate DCs with gamma-rays in a gamma-irradiation chamber (irradiation dose, 25 Gy). Irradiation will prevent the proliferation of DCs, which could otherwise give false results.Adjust concentration of lymphocytes to 1.0 x 106 cells/ml. Add 100 µl of cell suspension to irradiated DCs.Set positive controls by stimulating CD4+ T cells and CD8+ T cells with Concanavalin A (final concentration, 5 µg/ml).Keep plates at 37 °C in a humidified CO2 incubator. After 72 h, add 1.0 µCi 3H-thymidine per well of the plate set up with T cell proliferation assay. Keep plates back into the CO2 incubator. After 18 h, transfer plates to -20 °C. Plates can be thawed immediately or next day.Harvest the cells onto a filter paper and wash them using an automated cell harvester. Measure the 3H-thymidine levels on filter paper using a beta scintillation counter.Collect culture supernatants from plate set up with T cell polarization assay, after 96 h. Store supernatants at -80 °C or immediately analyze for TH1, TH2, TH17 signature cytokines by ELISA.RecipesRPMI-10RPMI-1640 base medium supplemented with 10% heat-inactivated FBS and 1% antibiotic-antimycotic solutionReferencesKumar, P., John, V., Marathe, S., Das, G. and Bhaskar, S. (2015). Mycobacterium indicus pranii induces dendritic cell activation, survival, and Th1/Th17 polarization potential in a TLR-dependent manner. J Leukoc Biol 97(3): 511-520. Muul, L. M., Silvin, C., James, S. P. and Candotti, F. (2008). Measurement of proliferative responses of cultured lymphocytes. Curr Protoc Immunol Chapter 7: Unit 7 10 11-17 10 24.How to cite this protocol: Kumar, P. and Bhaskar, S. (2016). Analysis of T Cell Proliferating and Polarizing Potential of Murine Dendritic Cells in Allogeneic-mixed Leukocyte Reaction. Bio-protocol 6(5): e1750. DOI: 10.21769/BioProtoc.1750; Full Text

5.树突状细胞（DC）疗法是什么原理？主要治疗什么？

树突状细胞免疫疗法（简称DC疗法，下文均称DC疗法）是机体内最有效、功能最强的专职抗原提呈细胞（APC）；2.它能高效地摄取、加工处理和递呈抗原；3.未成熟DC具有较强的捕获抗原及迁移能力；4.成熟DC能有效激活初始型T细胞；5.处于启动、调控、并维持免疫应答的中心环节。DC疗法的优势1.整个治疗过程病人简单易行，不需要采集大量的外周血;3.诱导并激活病人自身的特异性抗肿瘤免疫反应;

6.人体皮肤中的黑色素细胞是属于树突状细胞的一种吗

黑色素细胞是属于树突状细胞。黑色素细胞（melanophore）含有一种黑色素，称为皮肤黑色素（eumelanin），含有黑色素并分布在细胞中的囊泡，酪氨酸经过一系列的化学反应就会生成皮肤黑色素，将各种原料合成黑色素的关键酵素是酪胺酸酶，黑色素将无法被合成，有些两栖类的皮肤黑色素会带有其它的色素，例如有一种蛙类（phyllomedusine）的黑色素细胞中被发现出奇特的深红色色素。它累积在皮肤黑色素周围。不过大多数的黑色素细胞仍然被皮肤黑色素所独占，人类只拥有一种色素细胞，也就是哺乳类身上与黑色素细胞相对应的的黑素细胞。

7.树突状细胞的来源

人树突状细胞起源于造血干细胞(hemopoieticstemcell)。①髓样干细胞在GM-CSF的刺激下分化为DC，与单核细胞和粒细胞有共同的前体细胞；包括朗格汉斯细胞（Langerhans cells，间皮（或真皮）DCs以及单核细胞衍生的DCs等②来源于淋巴样干细胞，称为淋巴样DC(Lymophoid dendritic cells，LDC)或浆细胞样DC(plasmacytoid dendritic cells，与T细胞和NK细胞有共同的前体细胞。树突状细胞(DC)尽管数量不足外周血单核细胞的1%。