脂多糖诱导C57/BL6小鼠骨髓巨噬细胞表达共刺激分子CD80、CD86实验研究

Expression of costimulatory molecules CD80 and CD86 in bone marrow macrophages of C57/BL6 mice induced by lipopolysaccharide

目的:探究细菌脂多糖(LPS)诱导C57/BL6小鼠骨髓巨噬细胞表达共刺激分子CD80、CD86的情况。方法:从C57/BL6小鼠的股骨和胫骨中无菌分离骨髓细胞并使用25 ng/ml巨噬细胞集落刺激因子(M-CSF)进行刺激。通过换液对其进行纯化和扩增培养,经过7 d的分化培养和形态学观察,获得骨髓来源单核细胞。用异硫氰酸荧光素(FITC)标记抗小鼠F4/80抗体,用藻红蛋白(PE)标记抗小鼠CD11b抗体,流式细胞仪检测确定该细胞是否为小鼠骨髓巨噬细胞(BMDM)。观察组用LPS 1μg/ml刺激培养BMDM,对照组用含10%胎牛血清(FBS)的等量L-DMEM培养。培养24 h后用流式细胞仪检测FITC标记的抗CD80(B7-1)抗体和抗CD86(B7-2)抗体。结果:新分离的小鼠骨髓单核细胞呈小圆形,两端伸出触角,培养5 d后细胞呈椭圆形,两端的触角更明显,增殖依赖M-CSF。流式细胞仪检测结果显示,分离的小鼠骨髓单核细胞CD11b抗体阳性,纯度较高;LPS刺激后实验组较对照组表达共刺激分子CD80、CD86明显增多。结论:LPS刺激可使C57/BL6小鼠骨髓巨噬细胞表达共刺激分子CD80、CD86。

Objective:To explore expression of costimulatory molecules CD80 and CD86 in bone marrow macrophages of C57/BL6 mice induced by lipopolysaccharide.Methods:Bone marrow cells were isolated from femur and tibia of C57/BL6 mice and stimulated with 100 ng/ml macrophage colony stimulating factor(M-CSF).After 7 days of differentiation culture and morphological observation,bone marrow-derived monocytes were obtained.The anti mouse F4/80 antibody was labeled with fluorescein isothiocyanate(FITC),and anti mouse CD11b antibody was labeled with P-phycoerythrin(PE).Flow cytometry was used to determine whether the cells were bone marrow-derived macrophages(BMDM).The observation group was stimulated with LPS 1μg/ml,and the control group was cultured with equal amount of L-DMEM containing 10%fetal bovine serum(FBS).After 24 hours of culture,anti-mouse CD80(B7-1)and anti-mouse CD86(B7-2)antibody labeled with FITC were detected by flow cytometry.Results:The newly isolated mouse bone marrow mononuclear cells had a small round shape with tentacles at both ends.After 5 days of culture,the cells were elliptical and the tentacles at both ends were more obvious.The proliferation depends on M-CSF.The results of flow cytometry showed that CD11b antibody was positive and the purity was high;the expression of costimulatory molecules CD80 and CD86 in the experimental group was significantly higher than that in the control group after LPS stimulation.Conclusion:LPS can induce the expression of costimulatory molecules CD80 and CD86 in bone marrow macrophages of C57/BL6 mice.