小鼠骨髓源性巨噬细胞极化的体外诱导方法探究

目的:探究小鼠骨髓前体细胞体外诱导成为不同极化状态(M1和M2)巨噬细胞的优化方法。方法:健康C57BL/6小鼠麻醉处死,收集其股骨和胫骨腔内容物,经筛网过滤、红细胞裂解后,在RPMI-1640完全培养基中培养16h,收集未贴壁的骨髓前体细胞重新接种于6孔板。根据培养基中所加刺激剂的种类、剂量不同进行实验分组,于不同时点收集细胞,光镜下观察各组细胞形态学变化,流式细胞术及RT-qPCR检测不同极化状态巨噬细胞的相应标志物。结果:(1)小鼠骨髓前体细胞经50μg/L巨噬细胞集落刺激因子(M-CSF)刺激72 h后,CD11b阳染率达到90%以上;刺激96 h后,F4/80的阳染率达到95%以上。40μg/L的粒-巨噬细胞集落刺激因子(GM-CSF)刺激96 h后,CD11b阳染率也达到了90%以上,F4/80阳染率至144 h达到峰值(58.2%);(2)在M-CSF刺激所得单核-巨噬细胞的基础上,给予M1型巨噬细胞诱导剂(25μg/L LPS和10μg/L IFN-γ)刺激24 h,可见CD86的阳染率大于90%;给予M2型巨噬细胞诱导剂(20μg/L IL-4和IL-13)刺激后CD206的阳染率始终处于较低水平(10%左右);(3)在GM-CSF刺激的基础上,给予M1型巨噬细胞诱导剂刺激24 h,可见CD86的阳染率大于90%;而当细胞接受M2型巨噬细胞诱导剂刺激96 h,CD206的阳染率达68.98%;(4)RT-qPCR结果显示在给予相应极化诱导剂刺激后,M1型巨噬细胞标志物诱导型一氧化氮合酶(iNOS)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)、IL-12,以及M2型巨噬细胞标志物:几丁质酶3样蛋白3(Chi3l3/Ym1)、甘露糖受体(MR)和精氨酸酶1(Arg-1)的mRNA表达均明显高于对照组(P<0.01)。结论:(1)C57BL/6小鼠骨髓前体细胞受到M-CSF或GM-CSF诱导后90%以上细胞均可向单核细胞分化,M-CSF可诱导90%以上的细胞为成熟巨噬细胞,GM-CSF可诱导58%的细胞为成熟巨噬细胞;(2)在M-CSF前期诱导的基础上,联合LPS和IFN-γ易于诱导出M1型巨噬细胞,但联合IL-4和IL-13难以获得M2型巨噬细胞;(3)在GM-CSF前期诱导的基础上,联合LPS和IFN-γ易于诱导出M1型巨噬细胞,联合IL-4和IL-13也可将大部分细胞诱导成为M2型巨噬细胞。

Methods of in vitro induction of polarization from mouse bone marrow-derived macrophages

AIM:To explore the optimal method for isolation of mouse bone marrow precursor cells and induction into different polarization states(M1 and M2).METHODS:The contents of femur and tibia were collected from C57 BL/6 mice. After filtering through meshes and erythrocyte lysis,cells were cultured in RPMI-1640 complete medium for 16 h. Non-adherent cells were collected and re-seeded in culture plates. The cells were collected at different time points. The morphological changes of the cells were observed under light microscope. The corresponding markers of macrophages in different polarization states were detected by flow cytometry and RT-qPCR.RESULTS:(1)After stimulation with 50 μg/L M-CSF for 72 h,the positive staining rate of CD11 b was more than 90%. The positive staining rate of F4/80 was over 95% after stimulation for 96 h. After 96 h of stimulation with 40 μg/L GM-CSF,the positive staining rate of CD11 b also reached more than 90%. The positive staining rate of F4/80 reached the peak at 144 h(58. 2%).(2)On the basis of pre-induction of M-CSF,followed by 25 μg/L LPS and 10 μg/L IFN-γ stimulation for 24 h,the positive staining rate of CD86 was greater than 90%. Under the same circumstances,20 μg/L IL-4 and IL-13 were given at different time points which showed that the positive staining rate of CD206 was always low(<10%).(3)On the basis of pre-induction of GM-CSF,followed by 25 μg/L LPS and 10 ng/mL IFN-γ stimulation for 24 h,the positive staining rate of CD86 was greater than 90%. When the cells were stimulated with 20 μg/L IL-4 and IL-13 for 96 h,the positive staining rates of CD206 were 68. 98%.(4)RT-qPCR results showed that after simulation the mRNA expression of the markers for M1 macrophages(iNOS,IL-6,TNF-α and IL-12)and M2 macrophages(Ym1,MR and Arg-1)were significantly higher than that in the control group(P<0. 01).CONCLUSION:(1)Both M-CSF and GM-CSF induce more than 90% of bone marrow precursor cells from C57 BL/6 mouse into monocytes. 90% of bone marrow precursor cells differentiate to mature macrophages by M-CSF,and 58% of bone marrow precursor cells induced by GM-CSF become mature macrophages.(2)On the basis of pre-induction of M-CSF,LPS joint with IFN-γ is easy to induce M1 macrophages. However,it is difficult to obtain M2 macrophages by combination of IL-4 and IL-13.(3)On the basis of pre-induction of GM-CSF,LPS joint with IFN-γ is easy to induce M1 macrophages. Combined stimulation of IL-4 and IL-13 induces most cells to M2 macrophages.