| ObjectiveTo analyze the subsets of dendritic cells(DCs)in synovial fluid of patients with RA,in order to find out a new subsets of DC in inflammatory local microenvironment of patients with RA,and to analyze the effect of this subsets of DCs on the function of inflammatory cells in synovial local microenvironment,in order to provide new clues and theoretical support for the clinical treatment of rheumatoid arthritis.Methods1.Select the peripheral blood of 26 normal people from the Physical Examination Department of the First Affiliated Hospital of the Army Military Medical University as the healthy control group,and clinically use the peripheral blood of 60 patients with RA and 124 patients with RA who have been diagnosed in the Department of Rheumatology and Immunology of the First Affiliated Hospital of the Army Military Medical University.Membrane fluid is the experimental group,and RA patients meet the criteria for the diagnosis of RA in the"2018 Chinese Rheumatoid Arthritis Diagnosis and Treatment Guidelines";except for RA,other diseases that may affect the body’s immune response are excluded;2.The density gradient centrifugation method of human peripheral blood lymphocyte separation solution was used to separate and extract the mononuclear cells(Peripheral Blood Mononuclear Cells,PBMCs)from the peripheral blood of normal people and the peripheral blood of RA patients and the mononuclear cells from the synovial fluid of RA patients(Synovial Fluid Mononuclear Cells,SFMCs),flow cytometry detects the following DC subsets:plasma cell-like DC(Plasma dendritic cells,p DC,),myeloid DC(Myeloid dendritic cells,m DC,),CD1c~+DC and CD141~+DC;3.Flow cytometry was used to analyze the proportion of DC cell subsets in the peripheral blood of 26 normal people,the peripheral blood of 60 RA patients,and the synovial fluid of 124 RA patients.At the same time,the peripheral blood of normal people and the peripheral blood of RA patients were analyzed.The proportion of DC subsets in the peripheral blood of RA patients and the synovial fluid of RA patients were statistically compared;4.Using the biostatistical analysis software Graphpad Prism8 to analyze the correlation between the DC subsets in the peripheral blood and synovial fluid of RA patients and the clinical indicators of RA patients;5.Subsequently,RNA-seq data detection was performed on PBMC in peripheral blood and SFMC in synovial fluid of 6 patients with RA to analyze the expression of DC activation biomarkers and activated transcription pathway in synovial fluid of RA patients;look for RA patients New DC subsets existing in the membrane fluid;6.Expand the sample size and detect EBI3(Interleukin-27 subunit beta)expressed by DC in peripheral blood of 26 RA patients and synovial fluid of 24 RA patients by flow cytometry;7.The biostatistical analysis software Graphpad Prism8 was used to analyze the correlation between CD11c~+EBI3~+DC,CD1c~+EBI3~+DC,CD141~+EBI3~+DC subsets and clinical indicators in the peripheral blood and synovial fluid of RA patients;8.After that,we used EILSA experimental technology to detect the level of IL-27 in the peripheral blood supernatant of 41 normal people,the peripheral blood supernatant of 41RA patients,and the synovial fluid supernatant of 50 RA patients;9.Finally,we used the cytokine IL-27 to stimulate the inflammatory cells in the synovial fluid of RA patients,and the chemiluminescence immunoassay instrument detected the cytokine secretion in the supernatant of the inflammatory cells in the synovial fluid of the RA patients stimulated by IL-27.Results1.Compared with the healthy control group,the ratio of CD123~+DC and CD1c~+DC in the peripheral blood of RA patients decreased(P<0.0001,P=0.0098);while the ratio of CD11c~+DC and CD141~+DC was not significantly different(P=0.1088,P=4101).Compared with the synovial fluid of RA patients,the proportion of CD11c~+DC,CD1c~+DC and CD141~+DC in the synovial fluid of RA patients was significantly higher than that in the peripheral blood of RA patients(P<0.0001,P=0.0007,P<0.0001);while there is no significant difference in the ratio of CD123~+DC in the peripheral blood of RA patients and synovitis in RA patients(P=0.4399);2.The CD123~+DC,CD11c~+DC,CD1c~+DC,CD141~+DC in the peripheral blood and synovial fluid of RA patients are wirelessly correlated with RA clinical indicators DAS-28,RF,CCP,ESR.Peripheral blood CD123~+DC,CD11c~+DC,CD1c~+DC,CD141~+DC in RA patients and synovial fluid CD11c~+DC,CD1c~+DC,CD141~+DC in RA patients are wirelessly related to CRP,but synovial fluid CD123~+DC in RA patients and CRP were negatively correlated,the difference was statistically significant,P=0.0362,R~2=0.0393;3.Compared with the peripheral blood of RA patients,the expression of CD28 family costimulatory molecules,cell adhesion molecules,cytokine receptor interactions,and IFN-γsignaling pathway in the synovial fluid of RA patients are all up-regulated,and the results also show that NF-κB And LEF1 transcription pathway is up-regulated;4.In the synovial fluid of RA patients,there is a group of new DC subgroups(Activate DC)that are different from RA peripheral blood activation.The activated gene phenotypes include CCR7,LAMP3,CCL22,etc.;and mo DC,c DC1,c DC2 and p DC genes The phenotypic RA synovial fluid and peripheral blood have little difference;5.The proportions of CD11c~+EBI3~+DC,CD1c~+EBI3~+DC,and CD141~+EBI3~+DC in the peripheral blood and synovial fluid stimulation group of RA patients were higher than those in the non-stimulation group((P=0.0004,P<0.0001,P<0.0001;P<0.0001,P<0.0001,P<0.0001).In addition,compared with the peripheral blood stimulation group of RA patients,the ratio of CD11c~+EBI3~+DC and CD1c~+EBI3~+DC in the synovial fluid stimulation group of RA patients increased(P=0.0007,P=0.0005),and the ratio of CD141~+EBI3~+DC has no significant difference(P=0.2503);6.The CD11c~+EBI3~+DC,CD1c~+EBI3~+DC,CD141~+EBI3~+DC in the peripheral blood and synovial fluid of RA patients are wirelessly correlated with the RA clinical indicators RF,CCP,ESR,CRP.CD11c~+EBI3~+DC,CD1c~+EBI3~+DC,CD141~+EBI3~+DC in the synovial fluid of RA patients are not related to DAS-28,and CD11c~+EBI3~+DC,CD1c~+EBI3~+DC and DAS in the peripheral blood of RA patients 28 no correlation,but CD141~+EBI3~+DC in peripheral blood of RA patients was negatively correlated with DAS-28,the difference was statistically significant,P=0.0106,R~2=0.3263;7.The level of IL-27 in the synovial fluid supernatant of RA patients was significantly higher than that in the peripheral blood of healthy controls and RA patients(P<0.0001,P<0.0001),and the level of IL-27 in the peripheral blood supernatant of RA patients Higher than the healthy control group(P=0101);8.Preliminary experimental results show that the inflammatory cells in the synovial fluid of RA patients secrete cytokines IL-6,IL-8,and TNF-αunder the condition of IL-27stimulation for 72 hours.IL-8 is a sensitive indicator.However,IL-1,IL-2 and IL-10 have no obvious trends.Under other stimulation conditions(IL-12 stimulation,IL-12 and IL-27 co-stimulation,and 24-hour stimulation)there is no obvious phenomenon.After expanding the sample size,the results showed that the inflammatory cells in the synovial fluid of RA patients secreted cytokines IL-8 and TNF-αunder the condition of IL-27stimulation for 72 hours.The difference was statistically significant(P=0.0010).,P=0.0401).The secreted cytokine IL-6 had no significant difference compared with the blank control group(P=0.0638).Conclusion1.CD123~+p DC in the synovial fluid of RA patients is not directly involved in the inflammatory progression of RA,and CD11c~+DC plays an important role in the formation of local inflammation of the synovial membrane in RA patients;2.DCs in the synovial fluid of RA patients are highly activated and play an important role in the occurrence of local inflammation of the synovial fluid of RA patients;and there is a group of activated new DC subgroups in the synovial fluid of RA patients;3.EBI3(a subunit of IL-27)can be used as an important marker of inflammation in the synovial fluid of RA patients.It plays an important role in the inflammatory process of RA and is worthy of our in-depth study;4.The concentration of IL-27 in the synovial fluid supernatant of RA patients is significantly higher than that in the peripheral blood supernatant of RA patients and the supernatant of healthy controls,and IL-27 stimulates the secretion of inflammatory cells in the synovial fluid of RA patients Factors IL-8 and TNF-αsuggest that CD1c~+EBI3~+DC can be used as an important DC subsets in the process of local inflammation in RA. |
PDF Full Text Request