CAJ | 학술논문

目的：：建立小鼠骨髓源性树突状细胞(DCs)体外培养和扩增方法,为以DCs为靶点的免疫治疗研究提供基础。方法：从C57BL/6小鼠股骨和胫骨中提取骨髓细胞,以含重组小鼠粒细胞巨噬细胞集落刺激因子(GM-CSF,400 U/ml)和 IL-4(200 U/ml)的 RPMI 1640(含10%FBS)进行诱导培养,于培养第4天加入脂多糖(LPS)继续培养2 d。利用倒置相差显微镜和透射电子显微镜观察细胞生长状态和超微结构,流式细胞术鉴定培养第6天时DCs纯度及免疫表型,流式细胞微球捕获芯片技术检测第2、4、6天培养上清中细胞因子IL-6、IL-10、巨噬细胞趋化因子-1(MCP-1)、干扰素-1(IFN-1)、TNF-α、IL-12 p70含量。结果：重组小鼠 GM-CSF和 IL-4诱导培养小鼠骨髓细胞4 d,加入脂多糖(LPS)继续培养2 d,近90%细胞高表达DCs特征性标志物 CD11c,高表达抗原提呈分子 MHC-Ⅱ和共刺激分子CD86、CD80,具有典型的树突状形态学特征和分泌炎症性细胞因子 IL-6、IL-10、MCP-1、TNF-α和 IL-12p70的功能。结论：小鼠骨髓细胞以重组小鼠 GM-CSF和 IL-4体外诱导培养的 DCs具有较高的纯度,保持了体内的形态学、免疫表型及功能特征。
목적：：건립소서골수원성수돌상세포(DCs)체외배양화확증방법,위이DCs위파점적면역치료연구제공기출。방법：종C57BL/6소서고골화경골중제취골수세포,이함중조소서립세포거서세포집락자격인자(GM-CSF,400 U/ml)화 IL-4(200 U/ml)적 RPMI 1640(함10%FBS)진행유도배양,우배양제4천가입지다당(LPS)계속배양2 d。이용도치상차현미경화투사전자현미경관찰세포생장상태화초미결구,류식세포술감정배양제6천시DCs순도급면역표형,류식세포미구포획심편기술검측제2、4、6천배양상청중세포인자IL-6、IL-10、거서세포추화인자-1(MCP-1)、간우소-1(IFN-1)、TNF-α、IL-12 p70함량。결과：중조소서 GM-CSF화 IL-4유도배양소서골수세포4 d,가입지다당(LPS)계속배양2 d,근90%세포고표체DCs특정성표지물 CD11c,고표체항원제정분자 MHC-Ⅱ화공자격분자CD86、CD80,구유전형적수돌상형태학특정화분비염증성세포인자 IL-6、IL-10、MCP-1、TNF-α화 IL-12p70적공능。결론：소서골수세포이중조소서 GM-CSF화 IL-4체외유도배양적 DCs구유교고적순도,보지료체내적형태학、면역표형급공능특정。
Objective:To establish the method for cultivation and amplification of bone marrow-derived dendritic cells (DCs ) of mice in vitro, in order to provide an experimental base for the DC-targeting therapeutic study.Methods:Bone marrow cells of C57BL/6 mice were obtained and cultured in RPMI1640 containing recombi-nant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF)400 U/ml,interleukin-4 (IL-4)200 U/ml,and 10% fetal bovine serum invitro. On the 4th day,lipopolysaccharide (LPS)was added.Two days later, the morphology and ultrastucture of DCs were observed by phase contrast microscope and transmission electron microscope. The purity and immunophenotype of DCs were verified by flow cytometry on day 6. The contents of cytokines including IL-6,IL-10,monocyte chemotactic protein-1 (MCP-1),interferon-1 (IFN-1),tumor necrosis factor-α(TNF-α),IL-12p70 in supernatant were determined by cytometric bead array.Results:After cultured for 4 days followed by stimulation with LPS for 2 days,nearly 90% of mouse bone marrow cells expressed the charac-teristic DC marker-CD11c,with high level of antigen presentation molecule-major histocompability complex-Ⅱ(MHC-Ⅱ)and costimulatory molecules-CD86 and CD80. The cells possessed the typical morphologic feature of DCs and the function of secreting pro-inflammatory cytokines such as IL-6,IL-10,MCP-1,TNF-αand IL-12p70. Conclusions:Culturing invitro with GM-CSF and IL-4,bone marrow cells of mice can be induced to become DCs with high purity,with their characteristics of morphology,immunophenotype,and immunological functions.