采用流式细胞分选术区分中枢神经系统内小胶质细胞和浸润巨噬细胞

Distinguishing microglia and infiltrating macrophages in central nervous system by fluorescence-activated cell sorting

目的建立通过流式细胞分选术区分中枢神经系统内小胶质细胞和浸润巨噬细胞的方法。方法用成年C57BL/6小鼠建立双侧颈总动脉狭窄模型,分别采用集落刺激因子1受体抑制剂PLX5622或氯膦酸盐脂质体处理。将分离、匀浆、重悬后的小鼠脑和脊髓组织进行Percoll密度梯度离心,得到单核细胞悬液。采用CD45、CD11b和淋巴细胞抗原6家族成员C(Ly6C)抗体进行流式分选,获得小胶质细胞(CD11b^(+)CD45^(low)Ly6C^(-)细胞)和浸润巨噬细胞(CD11b^(+)CD45^(high)Ly6C^(+)细胞),并验证PLX5622和氯膦酸盐脂质体2种给药范式获得的处理效果。结果通过CD45、CD11b和Ly6C抗体可以有效区分中枢神经系统中小胶质细胞和浸润巨噬细胞。与对照组比较,PLX5622处理后小胶质细胞数量减少(P=0.001),而氯膦酸盐脂质体处理后浸润巨噬细胞数量减少(P<0.001)。结论所建立的流式细胞分选方法可有效区分中枢神经系统中小胶质细胞和浸润巨噬细胞,PLX5622和氯膦酸盐脂质体2种给药范式可分别选择性清除中枢神经系统内的小胶质细胞和浸润巨噬细胞。

Objective To establish a method for distinguishing microglia and infiltrating macrophages in central nervous system by fluorescence-activated cell sorting.Methods Adult C57BL/6 mice with bilateral common carotid artery stenosis were treated with PLX5622(a colony stimulating factor 1 receptor inhibitor)or clodronate liposomes.After separating,homogenizing and resuspending brain and spinal cord tissue,the mononuclear cell suspensions were obtained by Percoll density gradient centrifugation.Anti-CD45,anti-CD11b,and anti-lymphocyte antigen 6 family member C(Ly6C)were used to obtain microglia(CD11b^(+)CD45^(low)Ly6C^(-)cells)and infiltrating macrophages(CD11b^(+)CD45^(high)Ly6C^(+)cells)by fluorescence-activated cell sorting.The treatment effects of PLX5622 and clodronate liposomes were verified.Results Microglia and infiltrating macrophages in the central nervous system were effectively distinguished by anti-CD45,anti-CD11b,and anti-Ly6C.Compared with the control group,the number of microglia was significantly decreased after PLX5622 treatment(P=0.001),while the number of infiltrating macrophages was significantly decreased after clodronate liposome treatment(P<0.001).Conclusion The established method can effectively distinguish microglia and infiltrating macrophages in central nervous system by fluorescence-activated cell sorting,and PLX5622 and clodronate liposomes can selectively eliminate microglia and infiltrating macrophages in the central nervous system.