摘要
目的 研究经典活化型巨噬细胞（M1）对RA滑膜成纤维细胞（FLS）和对照组OA-FLS增殖的影响,为进一步研究巨噬细胞在RA疾病进展中的作用奠定基础.方法 采用脂多糖和IFN-γ体外诱导人单核细胞（THP-1）为M1,流式细胞术（FCM）检测M1表面特异性标志分子HLA-DR和CD197表达.transwell非接触式共培养M1和RA-FLS、OA-FLS,结晶紫染色法观察共培养48 h后RA-FLS、OA-FLS的增殖情况.MTS法检测M1分泌细胞因子对RA-FLS、OA-FLS增殖的影响.ELISA检测单独培养体系及共培养体系细胞培养上清中细胞因子TNF-α和IL-12的变化.采用配对t检验比较分析.结果 脂多糖和IFN-γ诱导THP-1为M1后,M1表面标记分子CD197和HLA-DR阳性率为78.25％和87.96％.RA-FLS、OA-FLS与M1共培养48 h后,RA-FLS、OA-FLS增殖受到明显抑制,共培养组显微镜下每视野RA-FLS、OA-FLS细胞数分别为（64±30）、（85 ±23）,RA-FLS、OA-FLS单独培养组分别为（467±87）、（263±78）,差异有统计学意义（t=7.459,3.791;P均＜0.05）.M1与FLS共培养对RA-FLS、OA-FLS的增殖有明显抑制作用,差异有统计学意义（t=-7.155,-8.111;P均＜0.05）.RA-FLS单独培养组和M1与RA-FLS共培养组培养上清中TNF-α的浓度分别是（0.024±0.011） ng/ml、（0.832±0.241） ng/ml,IL-12浓度分别为（0.033±0.015） ng/ml、（0.372±0.122） ng/ml;OA-FLS单独培养组和M1与OA-FLS共培养组培养上清中TNF-α的浓度分别是（0.031 ±0.017） ng/ml、（0.854±0.323） ng/ml,IL-12浓度分别为（0.012±0.009） ng/ml、（0.373±0.144） ng/ml;共培养组TNF-α和IL-12浓度均明显升高,差异均有统计学意义（t=-4.997,-4.777,-4.407,-4.334;P均＜0.05）,RA组与OA组的结果一致.结论 M1与RA-FLS、OA-FLS共培养后,显著抑制RA-FLS、OA-FLS增殖,可能与共培养上清中TNF-α和IL-12的增加有关.
Objective To investigate the influence of classically activated macrophage （M1） on the proliferation of rheumatoid arthritis （RA） fibroblast-like synovial （FLS） and osteoarthritis （OA） FLS proliferation.Methods Human monocytes leukemia cells （THP）-1 were induced into M1 by lipopolysaccharides （LPS） and interferon gamma （IFN-γ）,M1 specific surface molecular markers human leukocyte antigen （HLA）-DR and CD197 were detected by flow cytometry （FCM）.RA-FLS and OA-FLS were co-cultured with M1 by transwell chambers,the proliferation of RA-FLS and OA-FLS were observed by crystal violet staining assay.MTS was used to detect cytokines secreted from M1 on the multiplication of RA-FLS and OA-FLS.TNF-α and IL-12 were detected by enzyme linked immunosorbent assay （ELISA）.Paried student t test was used for statistical analysis.Results THP-1 were induced into M1 by LPS and IFN-γ,the expression rates of M1 surface specific molecular markers HLA-DR and CD197 were 78.25% and 87.96%.Crystal violet staining showed that RA-FLS and OA-FLS proliferation were significantly inhibited after co-cultured with M1 48 h,RA-FLS and OA-FLS of each vision under microscope in co-culture groups were （64 ±30） and （85 ±23） respectively,while the RA-FLS and OA-FLS in separate culture groups were （467±87） and （263±78） respectively,the difference was statistically significant （t=7.459,3.791;P〈0.05）.MTS assay indirectly reflected that the cytokines from M1 suppressed RA-FLS and OA-FLS proliferation （t=-7.155,-8.111;P〈0.05）.The concentration of TNF-α in cell culture supernatants secreted from RA-FLS group and RA-FLS/M1 co-culture group respectively were （0.024±0.01 1） ng/ml and （0.832±0.241） ng/ml respectively,the concentration of IL-12 from the two groups were （0.033±0.015） ng/ml and （0.372±0.122） ng/ml respectively.TNF-α from OA-FLS and OA-FLS/M1 co-culture group respectively were （0.031±0.017） ng/ml and （0.852±0.323） ng/ml,IL-12 were （0.012±0.009
出处
《中华风湿病学杂志》
CAS
CSCD
北大核心
2015年第8期540-544,I0001,共6页
Chinese Journal of Rheumatology
基金
国家自然科学基金面上项目（81373203）
国家自然科学基金青年项目（81302563）
上海市科委生物医药领域重点项目（11DZ1973802）,上海市浦东新区卫生局科技项目（PW2013B-5）
关键词
关节炎
类风湿
滑膜成纤维细胞
巨噬细胞
增殖
Arthritis rheumatoid
Fibroblast-like synovial
Macrophages
Proliferation
