CAJ | 학술논문

目的：建立小鼠骨髓源耐受性树突状细胞（CD11b＋F4/80＋TDCs）体外培养及功能鉴定方法。方法：以重组小鼠粒细胞-巨噬细胞集落刺激因子（ rmGM-CSF ）和重组白介素4（ rmIL-4）体外诱导小鼠骨髓细胞分化为未成熟树突细胞（iDCs），收集第六天的细胞采用流式细胞术分离纯化CD11b＋F4/80＋TDCs。倒置显微镜动态观察细胞形态学变化，流式细胞术观察并分选CD11b＋F4/80＋TDCs，CD11b＋F4/80＋TDCs的耐受功能通过MHCⅡ类分子、CD83、IDO、TLR-2的表达及IL-10和TGF-β1细胞因子的产生进行评价。流式细胞术测定MHCⅡ类分子表达，免疫组化法测定CD83、IDO、TLR-2的表达，ELISA法测定IL-10和TGF-β1的水平，同时设LPS刺激诱导的成熟树突细胞（mDCs）作对照。结果：镜下可见新鲜分离的骨髓细胞呈圆形，体积小，2 d后，部分细胞形态发生改变，体积增大，细胞聚集成团，培养5～6 d，细胞集落增多，形态不规则。同时表面出现较多毛刺样突起。 CD11b＋F4/80＋TDCs 含量在23％左右，经流式分选后细胞纯度达到99％以上。与 mDCs 比较， CD11b＋F4/80＋TDCs低表达MHCⅡ和CD83，高表达IDO、TLR-2，并分泌IL-10和TGF-β1。结论：用小鼠rmGM-CSF和rmIL-4体外能成功诱导小鼠骨髓源CD11b＋F4/80＋TDCs，并且CD11b＋F4/80＋TDCs通过表达IL-10、TGF-β1、IDO和TLR-2显示耐受功能。
목적：건립소서골수원내수성수돌상세포（CD11b＋F4/80＋TDCs）체외배양급공능감정방법。방법：이중조소서립세포-거서세포집락자격인자（ rmGM-CSF ）화중조백개소4（ rmIL-4）체외유도소서골수세포분화위미성숙수돌세포（iDCs），수집제륙천적세포채용류식세포술분리순화CD11b＋F4/80＋TDCs。도치현미경동태관찰세포형태학변화，류식세포술관찰병분선CD11b＋F4/80＋TDCs，CD11b＋F4/80＋TDCs적내수공능통과MHCⅡ류분자、CD83、IDO、TLR-2적표체급IL-10화TGF-β1세포인자적산생진행평개。류식세포술측정MHCⅡ류분자표체，면역조화법측정CD83、IDO、TLR-2적표체，ELISA법측정IL-10화TGF-β1적수평，동시설LPS자격유도적성숙수돌세포（mDCs）작대조。결과：경하가견신선분리적골수세포정원형，체적소，2 d후，부분세포형태발생개변，체적증대，세포취집성단，배양5～6 d，세포집락증다，형태불규칙。동시표면출현교다모자양돌기。 CD11b＋F4/80＋TDCs 함량재23％좌우，경류식분선후세포순도체도99％이상。여 mDCs 비교， CD11b＋F4/80＋TDCs저표체MHCⅡ화CD83，고표체IDO、TLR-2，병분비IL-10화TGF-β1。결론：용소서rmGM-CSF화rmIL-4체외능성공유도소서골수원CD11b＋F4/80＋TDCs，병차CD11b＋F4/80＋TDCs통과표체IL-10、TGF-β1、IDO화TLR-2현시내수공능。
Objective:To establish the methods of isolated culture and functional identification of mice bone marrow derived tolerogenic dendritic cells (CD11b+F4/80 +TDCs) in vitro.Methods: Mice bone marrow cells were isolated and cultured to obtain iDCs with the simulation of mouse rmGM-CSF and rmIL-4.CD11b+F4/80 +TDCs were purified by fluorescence-activated cell sorting on day 6.The morphological changes of TDCs were observed with the inverted microscope dynamically .The expression of CD11b+F4/80 +TDCs were analyzed by the flow cytometry .Tolerogenic function of CD11b+F4/80 +TDCs was evaluated by the expression of MHCⅡ, CD83, IDO, TLR-2, IL-10 and TGF-β1.The expression of MHCⅡ was analyzed by the flow cytometry , and the expression of CD83, IDO and TLR-2 were analyzed by immune-histochemistry.The levels of IL-10 and TGF-β1 in the supernatant of CD11b+F4/80 +TDC were analyzed by ELISA .Meanwhile mature DCs ( mDCs) induced by LPS were used as control .Results:The fresh isolated bone marrow cells look like round and small under microscope .After two days of culture , cells became big and formed into clusters . Five or six days later, cells clusters increased, and the morphology of cells became irregular .At the same time, more dendrite ap-peared on the surface of cells .The percentage of CD11b+F4/80 +TDCs induced by rmGM-CSF and rmIL-4 was about 23%, and the purity of the purified BM CD11b+F4/80 +iDC was about 99%.Compared with mDCs, CD11b+F4/80 +TDCs expressed low levels of MHCⅡand CD83 and high levels of IDO, TLR-2, IL-10 and TGF-β1.Conclusion:CD11b+F4/80 +TDCs derived from mouse bone marrow could be induced successfully by rmGM-CSF and rmIL-4 in vitro.CD11b+F4/80 +TDCs showed tolerogenic function by the expressions of IL-10, TGF-β1, IDO and TLR-2.