5-酮戊二酸(ALA)联合二价铁离子体外诱导小鼠骨髓来源调节性树突状细胞的培养及鉴定  

Culture and identification of regulatory dendritic cells from murine bone marrow induced by 5-Aminolevulinic acid combined with ferrous iron in vitro

作　　者：张琦 蔡松洁 刘亚婷 侯剑刚 Zhang Qi;Cai Songjie;Liu Yating;Hou Jiangang(Department of Dermatology, Huashan Hospital, Fudan University, Shanghai 200040, China)

机构地区：[1]复旦大学附属华山医院皮肤科,上海200040 [2]复旦大学附属华山医院泌尿外科,上海200040 [3]上海市皮肤病研究所,200040 [4]美国波士顿马萨诸塞州哈佛大学医学院伯列跟女子医院肾脏内科移植研究中心,02115

出　　处：《中华器官移植杂志》2018年第10期627-631,共5页Chinese Journal of Organ Transplantation

基　　金：国家自然科学基金(81771683,81202302,81671550).

摘　　要：目的探讨由5-酮戊二酸(ALA)联合二价铁离子(SFC)诱导调节性树突状细胞(DCreg)的方法并从其细胞形态、免疫表型、功能对其进行鉴定。方法无菌条件下取C57BL/6小鼠的骨髓细胞,按体外诱导培养的培养体系不同,将其分为2组:传统DC(conventional dendritic cell, DCcon)组,用含重组鼠粒-巨噬细胞集落刺激因子(rmGM-CSF)10ng/ml,重组鼠白细胞介素-4(rmIL-4)10 ng/mL的RPMI 1640培养基诱导培养7 d;ALA-DCreg组,用含rmGM-CSF 20 ng/ml,5-ALA 1mmol/L + SFC 0.5 mmol/L的RPMI 1640培养基培养7 d。显微镜下及细胞涂片观察细胞形态;通过流式细胞术检测细胞表面分子;通过体外混合淋巴细胞培养实验以及体内的细胞增殖实验来检测细胞功能。结果ALA-DCreg在形态上具有树突状细胞的特征;细胞表面高表达CD11b、CD11c双阳性分子,与DCcon一致;与DCcon具有同样高表达的第一信号MHCII分子,但第二信号分子CD40、CD80、CD86的表达显著低于DCcon;体内、外两种细胞增殖实验均显示ALA-DCreg具有抑制效应T细胞活化增殖的能力。结论ALA/SFC可成功诱导小鼠骨髓细胞向DCreg分化。Objective To explore a method to generate regulatory dendritic cells (DCregs) from murine bone marrow induced by 5-Aminolevulinic acid combined with ferrous iron (ALA/SFC). Methods Bone marrow cells were obtained from male C57BL/6 mice. To generate conventional DCs (BM-DCcons), the cells were cultured in RPMI-1640 medium supplemented with 10% FCS, 10 ng/mL GM-CSF, 10 ng/mL IL-4 for 7 days. The cells were collected for the analysis. To generate DCregs by ALA/SFC, the cells were cultured in RPMI-1640 medium supplemented with 10% FCS, 20 ng/mL GM-CSF, 5-ALA 1 mmol/L+ SFC 0.5 mmol/L for 7 days. The morphology of ALA-DCregs was observed by microscope and cytospin with May-Grunwald-Giemsa stain. The surface markers of ALA-DCregs were observed by FACS. The function of ALA-DCregs was detected by in vitro mixed lymphocyte reaction (MLR) and in vivo lymphocyte proliferation assay. Results The generated ALA-DCregs displayed an irregular shape with areas of protrusion and demonstrated higher CD11b/CD11c and higher MHC-II but lower CD40, CD80, CD86 expression levels than DCcons. They also had immune regulation effects in both in vitro and in vivo lymphocyte proliferation assay. Conclusion This study illustrated a feasible approach for generating functional DCregs from murine bone marrow induced by ALA/SFC. These cells can be useful for research and application of DC immunotherapy in the future.

关 键 词：5-酮戊二酸 树突状细胞 小鼠 

分 类 号：R392.4[医药卫生—免疫学]