中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2013年
7期
488-494
,共7页
Gpnmb%骨髓源性巨噬细胞%M1型巨噬细胞%M2型巨噬细胞%MMR
Gpnmb%骨髓源性巨噬細胞%M1型巨噬細胞%M2型巨噬細胞%MMR
Gpnmb%골수원성거서세포%M1형거서세포%M2형거서세포%MMR
Gpnmb%Bone marrow-derived macrophages%M1 macrophages%M2 macrophages%MMR
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、iNOS mRNA在M1型BMMφs中高表达（P均＜0．01），而MMR、Arg-1 mRNA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Gpnmb mRNA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。
目的瞭解小鼠M1和M2型骨髓源性巨噬細胞（ BMMφs）中非轉移性黑色素瘤糖蛋白b（ Gpnmb）錶達的差異。方法原代培養小鼠BMMφs，免疫熒光染色F4/80和流式細胞儀檢測CD11b鑒定巨噬細胞；用IFN-γ和LPS誘導BMMφs嚮M1型分化，用IL-4誘導嚮M2型分化。實時熒光定量PCR檢測M1型巨噬細胞標誌物（ TNF-α、iNOS）、M2型巨噬細胞標誌物（ MMR、Arg-1）和Gpn-mb的mRNA錶達；免疫熒光雙染色、Western印跡、流式細胞儀檢測Gpnmb與MMR的蛋白錶達。結果（1）免疫熒光染色結果示BMMφs中F4/80高錶達；流式細胞儀檢測結果示BMMφs中有（92．7±6．1）％細胞錶達CD11b，提示BMMφs培養成功；（2）相對于未分化的M0型BMMφs，TNF-α、iNOS mRNA在M1型BMMφs中高錶達（P均＜0．01），而MMR、Arg-1 mRNA在M2型BMMφs中高錶達（P均＜0．01），提示原代M1、M2型BMMφs分化成功；（3）M2型BMMφs 的Gpnmb mRNA和蛋白錶達均較M0型和M1型BMMφs顯著增高（P均＜0．01）；免疫熒光雙染色及流式細胞儀結果顯示，BMMφs中Gpnmb與MMR共錶達，在M2型BMMφs中MMR暘性BMMφs有（83．2±9．7）％錶達Gpnmb。結論 M2型BMMφs的Gpnmb錶達較M1型BMMφs顯著增高，提示Gpnmb可能作為鑒彆M1、M2型巨噬細胞的標誌物，在巨噬細胞的錶型分化中起作用。
목적료해소서M1화M2형골수원성거서세포（ BMMφs）중비전이성흑색소류당단백b（ Gpnmb）표체적차이。방법원대배양소서BMMφs，면역형광염색F4/80화류식세포의검측CD11b감정거서세포；용IFN-γ화LPS유도BMMφs향M1형분화，용IL-4유도향M2형분화。실시형광정량PCR검측M1형거서세포표지물（ TNF-α、iNOS）、M2형거서세포표지물（ MMR、Arg-1）화Gpn-mb적mRNA표체；면역형광쌍염색、Western인적、류식세포의검측Gpnmb여MMR적단백표체。결과（1）면역형광염색결과시BMMφs중F4/80고표체；류식세포의검측결과시BMMφs중유（92．7±6．1）％세포표체CD11b，제시BMMφs배양성공；（2）상대우미분화적M0형BMMφs，TNF-α、iNOS mRNA재M1형BMMφs중고표체（P균＜0．01），이MMR、Arg-1 mRNA재M2형BMMφs중고표체（P균＜0．01），제시원대M1、M2형BMMφs분화성공；（3）M2형BMMφs 적Gpnmb mRNA화단백표체균교M0형화M1형BMMφs현저증고（P균＜0．01）；면역형광쌍염색급류식세포의결과현시，BMMφs중Gpnmb여MMR공표체，재M2형BMMφs중MMR양성BMMφs유（83．2±9．7）％표체Gpnmb。결론 M2형BMMφs적Gpnmb표체교M1형BMMφs현저증고，제시Gpnmb가능작위감별M1、M2형거서세포적표지물，재거서세포적표형분화중기작용。
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 immunofluorescence staining for F 4/80 and flow cytometry testing of CD11b.Interferon-γand lipopolysaccharide were used to induce differentiation of BMMφs towards M1 macrophages and interleukin-4 was adopted to induce differentiation of M 2 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 F 4/80 in BMMφs and flow cytometry results showed that CD11b was expressed in 92.7%±6.1% of BMMφs, suggesting that primary BMMφs were successfully cultured.(2) Compared with M0 BMMφs, mRNAs of TNF-αand 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 M 2 macrophages than that in M 1 macrophages in vitro, indicating that Gpnmb which takes part in the differentiation of macrophages might be used as a marker for identification of M 1 and M2 macrophages .
