CAJ | 학술논문

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.
목적 연구경전활화형거서세포(M1)대RA활막성섬유세포(FLS)화대조조OA-FLS증식적영향,위진일보연구거서세포재RA질병진전중적작용전정기출.방법 채용지다당화IFN-γ체외유도인단핵세포(THP-1)위M1,류식세포술(FCM)검측M1표면특이성표지분자HLA-DR화CD197표체.transwell비접촉식공배양M1화RA-FLS、OA-FLS,결정자염색법관찰공배양48 h후RA-FLS、OA-FLS적증식정황.MTS법검측M1분비세포인자대RA-FLS、OA-FLS증식적영향.ELISA검측단독배양체계급공배양체계세포배양상청중세포인자TNF-α화IL-12적변화.채용배대t검험비교분석.결과 지다당화IFN-γ유도THP-1위M1후,M1표면표기분자CD197화HLA-DR양성솔위78.25％화87.96％.RA-FLS、OA-FLS여M1공배양48 h후,RA-FLS、OA-FLS증식수도명현억제,공배양조현미경하매시야RA-FLS、OA-FLS세포수분별위(64±30)、(85 ±23),RA-FLS、OA-FLS단독배양조분별위(467±87)、(263±78),차이유통계학의의(t=7.459,3.791;P균＜0.05).M1여FLS공배양대RA-FLS、OA-FLS적증식유명현억제작용,차이유통계학의의(t=-7.155,-8.111;P균＜0.05).RA-FLS단독배양조화M1여RA-FLS공배양조배양상청중TNF-α적농도분별시(0.024±0.011) ng/ml、(0.832±0.241) ng/ml,IL-12농도분별위(0.033±0.015) ng/ml、(0.372±0.122) ng/ml;OA-FLS단독배양조화M1여OA-FLS공배양조배양상청중TNF-α적농도분별시(0.031 ±0.017) ng/ml、(0.854±0.323) ng/ml,IL-12농도분별위(0.012±0.009) ng/ml、(0.373±0.144) ng/ml;공배양조TNF-α화IL-12농도균명현승고,차이균유통계학의의(t=-4.997,-4.777,-4.407,-4.334;P균＜0.05),RA조여OA조적결과일치.결론 M1여RA-FLS、OA-FLS공배양후,현저억제RA-FLS、OA-FLS증식,가능여공배양상청중TNF-α화IL-12적증가유관.
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) ng/ml,(0.373±0.144) ng/ml.Compared with FLS separate culture group,the concentration of TNF-α and IL-12 were obviously elevated (t=-4.997,-4.777,-4.407,-4.334;P were all ＜0.05).Conclusion M1 can significantly inhibite RA-FLS and OA-FLS proliferation,this may be related to the increased concentration of TNF-α and IL-1 β in from cell culture supernatant.