摘要
初步研究IL-34对类风湿关节炎(rheumatoid arthritis,RA)滑膜间充质干细胞(synovial mesenchymal stem cell,SMSC)色素上皮衍生因子(pigment epithelium-derived factor,PEDF)表达的影响。选取6例RA患者,分离并培养其SMSC,加入PE标记的抗CD73、CD90、CD105抗体和APC标记的抗CD14、CD34、CD45抗体及同型对照,采用流式细胞术检测SMSC表型。而后加入IL-34(100ng/mL)刺激0h、6h、24h、48h以及72h、信号通路抑制剂(10μmol/L)刺激1h/IL-34(100ng/mL)刺激24h,通过ELISA检测SMSC培养上清中PEDF蛋白的表达,RT-PCR检测SMSC PEDF的转录水平。组间比较采用独立样本t检验。对培养细胞克隆进行鉴定,结果显示细胞表面阳性表达CD73、CD90、CD105,阴性表达CD14、CD34、CD45。PCR结果显示,与对照组相比,IL-34刺激6h的PEDF mRNA水平明显降低。同时ELISA表明,IL-34刺激24h、48h以及72h后的PEDF蛋白水平与对照组相比,也明显降低(P<0.05)。在细胞培养体系中加入NF-κB、p38MAPK信号通路抑制剂后,RA SMSC产生的PEDF水平明显升高(P<0.05)。以上结果显示,实验中分离纯化的RA SMSC高表达CD73、CD90、CD105,不表达CD14、CD34、CD45,符合SMSC表型特点。IL-34介导的抑制RA SMSC分泌PEDF可能通过激活p38 MAPK和NF-κB信号通路实现。
To investigate the effect of interleukin 34（IL-34）on the secretion of pigment epithelium derived-factor（PEDF）by synovial mesenchymal stem cells（SMSC）in patients with rheumatoid arthritis（RA）,RA SMSC were isolated from 6 RA patients and cultured in vitro.And then PE labeled anti-CD73,anti-CD90,anti-CD105 antibodies and APC labeled anti-CD14,anti-CD34,anti-CD45 antibodies and isotype control were added in the cell suspensions.SMSC phenotypes were detected by flow cytometry.RA SMSCs were stimulated with IL-34（100 ng/mL）or signaling pathway inhibitors（10μmol/L）/IL-34（100 ng/mL）.Enzyme linked immunosorbent assay（ELISA）was used to detect the level of PEDF in the cell supernatants,and reverse transcription polymerase chain reaction（RT-PCR）was used to determine the expression of PEDF mRNA.Statistical analysis was performed by t test.The results showed,1）CD73,CD90 and CD105 were strongly expressed on SMSC,and by contrast,CD14,CD34 and CD45 were almost not expressed.2）Compared with unstimulated RA SMSC,IL-34-stimulated RA SMSC showed lower expression of PEDF mRNA and protein significantly（P〈0.05）.Moreover,in the presence of SB203580 and IKK-16,the production of PEDF in the IL-34-stimulated RA SMSC was significantly increased（P〈0.05）.In conclusion,SMSC isolated from RA patients expressed higher CD73,CD90 and CD105 whereas lower CD14,CD34 and CD45 molecules,this is consistent with the phenotypic features of MSC.Consequently,IL-34 might be involved in the pathogenesis of RA by inhibiting SMSC＇secretion of PEDF through p38 MAPK and NF-κB signaling pathway.
作者
李寒
欧阳寻丽
汤亚微
张彦
李霞
王冰
LI Han1 , OU YANG Xun-li1, TANG Ya-wei1 , ZHANG Yan2 , LI Xia1, WANG Bing1(1. Department of Immunology, College of Basic Medical Science, Dalian Medical University, Dalian 116044, China; 2. Department of Rheumatology and Immunology, the Second Hospital of Dalian Medical University, Dalian 116023, Chin)
出处
《现代免疫学》
CAS
CSCD
北大核心
2018年第2期95-99,共5页
Current Immunology
基金
国家自然科学基金面上项目(81373214
81671606)
大连医科大学转化医学专项基金资助项目(2015010)
大连医科大学基础医学学科"青年教师助飞计划"项目
