Gpnmb在小鼠M1和M2型骨髓源性巨噬细胞中表达的差异

Differences of Gpnmb expression in M1 and M2 bone marrow-derived macrophages in mouse

目的了解小鼠M1和M2型骨髓源性巨噬细胞（BMMφs）中非转移性黑色素瘤糖蛋白b（Gpnmb）表达的差异。方法原代培养小鼠BMMφs，免疫荧光染色F4／80和流式细胞仪检测CD11b鉴定巨噬细胞；用IFN-γ和LPS诱导BMMφs向M1型分化，用IL-4诱导向M2型分化。实时荧光定量PCR检测M1型巨噬细胞标志物（TNF-α、iNOS）、M2型巨噬细胞标志物（MMR、Arg-1）和Gpn-mb的mRNA表达；免疫荧光双染色、Western印迹、流式细胞仪检测Gpnmb与MMR的蛋白表达。结果（1）免疫荧光染色结果示BMMφs中F4／80高表达；流式细胞仪检测结果示BMMφs中有（92．7±6．1）％细胞表达CD11b，提示BMMφs培养成功；（2）相对于未分化的M0型BMMφs，TNF-α、iNOSmRNA在M1型BMMφs中高表达（P均〈0．01），而MMR、Arg-1mRNA在M2型BMMφs中高表达（P均〈0．01），提示原代M1、M2型BMMφs分化成功；（3）M2型BMMφs的GpnmbmRNA和蛋白表达均较M0型和M1型BMMφs显著增高（P均〈0．01）；免疫荧光双染色及流式细胞仪结果显示，BMMφs中Gpnmb与MMR共表达，在M2型BMMφs中MMR阳性BMMφs有（83．2±9．7）％表达Gpnmb。结论M2型BMMφs的Gpnmb表达较M1型BMMφs显著增高，提示Gpnmb可能作为鉴别M1、M2型巨噬细胞的标志物，在巨噬细胞的表型分化中起作用。

Objective To investigate the differences of glycoprotein non-metastatic melanoma b （Gpnmb） expression between M1 and M2 bone marrow-derived macrophages （BMMφs） in mouse. Meth- ods Primary BMMφs were cultured and then identified by immunofluorescenee staining for F4/80 and flow cytometry testing of CDllb. Interferon-γ and lipopolysaccharide were used to induce differentiation of BMMφ towards M1 macrophages and interleukin-4 was adopted to induce differentiation of M2 macropha- ges. Realtime PCR was performed to analyze mRNA expressions of tumor necrosis factor （TNF-α）, induc- ible NO synthase （ iNOS）, macrophage mannose receptor （MMR）, arginase-1 （ Arg-1 ） and Gpnmb. Pro- teins of Gpnmb and MMR were detected by double immunofluorescence staining, Western blot and flow cy- tometry. Results （1） Immunofluorescence staining showed high expression of F4/80 in BMMφs and flow cytometry results showed that CDllb was expressed in 92.7% ±6.1% of BMMφs, suggesting that primary BMMφs were successfully cultured. （2） Compared with M0 BMMφs, mRNAs of TNF-α and iNOS were highly up-regulated in M1 BMMφs （ both P〈0. 01 ）, and mRNAs of MMR and Arg-1 were highly up-regula- ted in M2 BMMφs （ both P〈0.01 ）, indicating that differentiation of BMMφs towards M1 and M2 BMMφs were successfully induced. （3） Expressions of Gpnmb mRNA and Gpnmb protein were predominantly up- regulated in M2 BMMφs in comparison with those in M0 and M1 BMMφs （ both P〈0. 01 ）. Gpnmb and MMR were co-expressed in M2 BMMφs and 83.2% ±9.7% of MMR positive BMMφs expressed Gpnmb. Conclusion Gpnmb expression is significantly increased in M2 macrophages than that in M1 macrophages in vitro, indicating that Gpnmb which takes part in the differentiation of macrophages might be used as a marker for identification of M1 and M2 macrophages.