摘要
目的:研究小鼠脊髓损伤(SCI)急性期损伤区巨噬细胞表型和功能异质性。方法:将6~8周C57BL/6J雄鼠随机分为正常(normal)组和脊髓损伤(SCI)组,SCI组利用改造的Dumont系结镊建立标准脊髓钳夹损伤模型。在SCI急性期(1、3和7 d),利用流式细胞术检测脊髓损伤区中免疫细胞亚群的比例变化;培养原代小胶质细胞(MG)和骨髓来源巨噬细胞(BMDM),利用流式细胞术比较其增殖和吞噬功能差异;利用real time RT-PCR比较其免疫应答差异。结果:在小鼠SCI急性期,脊髓损伤区周围中性粒细胞比例短暂升高,损伤后第3 d恢复至正常水平;高表达CX3CR1的MG和高表达Ly6C的BMDM比例在损伤第1 d大量增加,在第3 d时达到峰值,之后在第7 d时呈现下降的趋势;在体外实验中,原代培养的MG比BMDM具有更强的增殖、吞噬能力;然而,在LPS 100 ng/ml和IFN-γ20 ng/ml细胞因子刺激下,BMDM比MG分泌较多的促炎作用的分子:iNOS、TNF-α和IL-1β;在IL-420 ng/ml细胞因子的作用下,BMDM比MG分泌较少的抗炎作用分子Arg1、Mcr1和YM1。结论:在小鼠SCI急性期,脊髓损伤区中存在两种来源不同的巨噬细胞,即中枢MG和外周招募而来的BMDM,并且MG增加的速度和数量显著快于和多于BMDM。体外实验中,发现MG比BMDM具有更强的增殖、吞噬能力;在相同炎性细胞因子的刺激下,MG分泌更多的抗炎细胞因子和较少的促炎细胞因子。
Objective:To study the macrophage heterogeneity such as phenotype and function during acute spinal cord injury(SCI)in mice.Methods:The C57BL/6J mouse were randomly divided into normal group and SCI group.The SCI animal model was established using modified Dumont tying forceps Dumont tying forceps.Fluorescence activated cell sorter(FACS)was used to detect the main subsets of immune inflammatory cells in the injured spinal cord at different time after spinal cord injury(1,3,and day 7);primary microglia(MG)and bone marrow-derived monocytes/macrophages(BMDM)were cultured in vitro,FACS was used to detect the heterogeneity of MG and BMDM such as proliferation,phagocytosis;The polarization level of MG and BMDM was analyzed by real-time RT-PCR.Results:In the acute phase of murine SCI,peripheral neutrophils in the injured area increased immediately,and returned to the normal homeostasis level on D3.MG with high expression of CX3 CR1 and BMDM with high expression of Ly6C were significantly increased on D1 after SCI,reached to the peak on D3,and then inclined to decrease on D7.Primary cultured MG showed significant stronger ability of proliferation and phagocytosis compared with BMDM.However,with the LPS(100 ng/ml)and IFN-γ(20 ng/ml)stimulation,more production of pro-inflammatory cytokines:iNOS,TNF-αand IL-1β;and with the IL-4(20 ng/ml)stimulation,more production of pro-inflammatory cytokines:Arg1,Mcr1 and YM1 were secreted by BMDM than those by MG.Conclusion:During acute phase in SCI,there are two origin of macrophages in injured spinal cord,one is tissue-resident MG originated from embryonic phase,anther is BMDM originated from adult phase,and the increase rate and quantity of MG was significantly faster and higher than that of BMDM.Therein,MG exhibit more strong ability of proliferation and phagocytosis,and under the same condition of inflammatory cytokines stimulation,MG produce much more anti-inflammation cytokines and less pro-inflammation cytokines.
作者
李鹏辉
梁世倩
胡昳旸
秦鸿雁
王哲
Li Penghui;Liang Shiqian;Hu Yiyang;Qin Hongyan;Wang Zhe(Department of Orthopaedics,Xijing Hospital,School of Basic Medicine,Air Force Medical University,Xi'an 710032,China;Department of Medical Genetics and Developmental Biology,School of Basic Medicine,Air Force Medical University,Xi'an 710032,China)
出处
《神经解剖学杂志》
CAS
CSCD
2021年第3期265-273,共9页
Chinese Journal of Neuroanatomy
基金
国家自然科学基金重点项目(81530018)
国家自然科学基金(81402438)
陕西省自然科学基金(2020JM-334)。
