小鼠骨髓源性巨噬细胞诱导及冻存方法的优化  

Optimization of induction and cryopreservation methods for mouse bone marrow-derived macrophages

作　　者：魏琼 赵梦竹 程序 刘梦华 张冬梅[1] WEI Qiong;ZHAO Mengzhu;CHENG Xu;LIU Menghua;ZHANG Dongmei(Dongzhimen Hospital,Beijing University of Chinese Medicine,Beijing 100700,China;Department of Traditional Chinese Medicine,Navy Medical University,Shanghai 200433,China;Kaifeng Traditional Chinese Medicine Hospital,Kaifeng 475000,China)

机构地区：[1]北京中医药大学东直门医院,北京100700 [2]海军军医大学中医系,上海200433 [3]开封市中医院,河南开封475000

出　　处：《中国病理生理杂志》2025年第3期611-618,共8页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.82474445);北京中医药大学“揭榜挂帅”项目(No.2022-JYB-JBZR-013)。

摘　　要：目的:探讨小鼠骨髓源性巨噬细胞(bone marrow-derived macrophages,BMDMs)诱导及冻存的适宜方法。方法:将小鼠成纤维细胞(L929细胞)于不同温度、接种密度、血清浓度培养条件下培养,ELISA法检测细胞上清中巨噬细胞集落刺激因子(macrophage colony-stimulating factor,M-CSF)浓度;提取小鼠骨髓细胞,细胞差速贴壁法将骨髓细胞预培养4 h,流式细胞术检测骨髓原驻留巨噬细胞比例;使用L929细胞上清或重组M-CSF诱导细胞7 d,倒置显微镜下观察细胞形态变化,流式细胞术检测诱导后细胞F4/80及CD11b双阳性占比;中性红法及ELISA法检测C57BL/6N和C57BL/6J两种亚型来源BMDMs吞噬能力和炎症反应水平;将提取的骨髓细胞即刻冻存后复苏再诱导,或将已成功诱导的BMDMs冻存后复苏,倒置显微镜下观察细胞形态,CCK-8法检测细胞活力,中性红法检测吞噬能力。结果:33℃、10%胎牛血清(fetal bovine serum,FBS)培养7 d条件下的L929细胞上清中富集高浓度M-CSF;骨髓细胞经预培养4 h,贴壁细胞中F4/80+占比显著高于悬浮细胞(P<0.01);经L929上清或重组M-CSF诱导7 d后细胞F4/80+CD11b+占比无显著差异(P>0.05);与C57BL/6J亚型来源BMDMs相比,C57BL/6N亚型来源BMDMs具有更强的吞噬能力(P<0.01),释放较低水平TNF-α(P<0.01)和IL-6(P<0.05),以及较高水平IL-1β(P<0.05);相较于将已成功诱导的BMDMs冻存后复苏,骨髓细胞提取后即刻冻存、复苏再诱导的BMDMs具有更好的巨噬细胞形态,更高的细胞活力(P<0.01)和更强的吞噬能力(P<0.01)。结论:通过33℃、10%FBS培养7 d的L929细胞上清富含更高浓度M-CSF,可成功诱导骨髓细胞分化为小鼠骨髓源性巨噬细胞,细胞差速贴壁法预培养可去除原骨髓驻留巨噬细胞,C57BL/6N和C57BL/6J两种亚型来源BMDMs吞噬能力和炎症反应水平不同,将提取的骨髓细胞即刻冻存后复苏再诱导是较好的BMDMs冻存方法。AIM:To explore suitable methods for the induction and cryopreservation of mouse bone marrow-derived macrophages(BMDMs).METHODS:Mouse fibroblasts(L929 cells)were cultured under varying conditions of temperature,seeding density,and serum concentration.The concentration of macrophage colony-stimulating factor(M-CSF)in the cell supernatant was measured using ELISA to determine the optimal conditions.Mouse bone marrow cells were extracted,and a differential adherence method was employed to preculture the bone marrow cells for 4 h,followed by flow cytometry to assess the proportion of resident macrophages.Flow cytometry was utilized to assess the ratio of F4/80 positive cells among the suspended and adherent cells.Induction of BMDMs was conducted using L929 cell supernatant or recombinant M-CSF for 7 d,and flow cytometry was applied to evaluate the proportion of F4/80 and CD11b double-positive cells.The morphologic changes during cell induction were observed under an inverted microscope,and the phagocytic capacity and inflammatory response levels of BMDMs derived from C57BL/6N and C57BL/6J mice were evaluated using neu-tral red and ELISA methods.The cells were immediately cryopreserved after extraction,and then induced after recovery,or cryopreserved after successful induction and recovered.The cell morphology was observed under an inverted micro-scope,cell viability was assessed using the CCK-8 method,and phagocytic ability was measured using the neutral red method.RESULTS:The M-CSF concentration in the supernatant from L929 cells cultured at 33℃,10%fetal bovine se-rum(FBS)for 7 d was rich.Following 4 h of pre-culture,the proportion of F4/80 positive cells in adherent cells was sig-nificantly higher than that in suspended cells(P<0.01).After 7 d of induction with L929 cell supernatant or recombinant M-CSF,the proportions of F4/80+CD11b+cells showed no significant difference(P>0.05).Compared with the BMDMs derived from C57BL/6J mice,those from C57BL/6N mice exhibited stronger phagocytic capacity(P<0.01),and released

关 键 词：小鼠骨髓源性巨噬细胞 原代细胞 成纤维细胞 巨噬细胞集落刺激因子 

分 类 号：R363[医药卫生—病理学] Q813.1[医药卫生—基础医学] Q952.5[生物学—生物工程]