人脐带间充质干细胞外泌体促进类风湿关节炎滑膜成纤维细胞凋亡的作用机制  

Mechanism of human umbilical cord mesenchymal stem cell derived exosomes in promoting apoptosis of synovial fibroblasts in rheumatoid arthritis

作　　者：柏林昆 苏雅珍 张明会 刘倩茹 张成强[2] 张莉芸[1] 张改连[2] Bai Linkun;Su Yazhen;Zhang Minghui;Liu Qianru;Zhang Chengqiang;Zhang Liyun;Zhang Gailian(Department of Rheumatology and Immunology,Third Hospital of Shanxi Medical University(Shanxi Bethune Hospital,Shanxi Academy of Medical Sciences,Tongji Shanxi Hospital),Taiyuan 030032,China;Department of Rheumatology and Immunology,Fifth Hospital of Shanxi Medical University,Shanxi Provincial People's Hospital,Taiyuan 030012,China)

机构地区：[1]山西医科大学第三医院(山西白求恩医院,山西医学科学院,同济山西医院)风湿免疫科,太原030032 [2]山西医科大学第五医院、山西省人民医院风湿免疫科,太原030012

出　　处：《中华风湿病学杂志》2024年第11期819-828,I0003,共11页Chinese Journal of Rheumatology

基　　金：山西省留学人员科技活动择优资助项目(20210003);山西省基础研究计划资助项目(202203021221266)。

摘　　要：目的探讨人脐带间充质干细胞外泌体(hUCMSC-Exos)对类风湿关节炎(RA)滑膜成纤维细胞(FLSs)不同亚型组蛋白去乙酰化酶(HDAC)表达水平的影响,阐明hUCMSC-Exos通过调控HDAC影响类风湿关节炎(RA)FLSs凋亡的可能作用机制。方法分离培养并鉴定hUCMSCs及hUCMSC-Exos。实时荧光定量PCR(RT-qPCR)检测hUCMSC-Exos干预后FLSs中HDAC mRNA表达水平的变化,筛选出受影响最大的HDAC类型。免疫印迹法(Western Blot)检测空白对照组、hUCMSC组、hUCMSC-Exos组、曲古抑菌素A(Trichostatin A,TSA)组和HDAC1抑制剂(Pyroxamide)组中FLS HDAC1蛋白水平和NF-κB p65、磷酸化-NF-κB p65(Ser 536)的表达水平,明确hUCMSC-Exos对FLSs中HDAC表达和NF-κB活性的影响。流式细胞术检测hUCMSC-Exos对FLSs凋亡的影响。ELISA检测hUCMSC-Exos对FLSs分泌TNF-α、IL-6、IL-1β、IL-8的影响。利用流式细胞术及ELISA检测空白对照组、NF-κB抑制剂(吡咯烷二硫代氨基甲酸盐(PDTC)组、hUCMSC-Exos组和PDTC+hUCMSC-Exos共同干预组FLSs的细胞凋亡水平及促炎细胞因子的分泌水平,进一步探究抑制NF-κB是否影响hUCMSC-Exos对RA FLSs的调节作用。将所有符合正态分布的实验数据,采用独立样本t检验或单因素方差分析比较组间差异,进一步两两比较采用LSD-t检验。结果培养的原代hUCMSC为贴壁生长的纺锤型细胞,hUCMSC-Exos为茶托状膜性囊泡,均符合鉴定标准。hUCMSC-Exos可降低RA FLSs中HDCA1 mRNA[(0.932±0.091),t=2.19,P<0.001]和蛋白[(0.204±0.012),t=8.28,P<0.001]的表达水平,且抑制作用较hUCMSC(t=1.06,P=0.009)和HDAC1抑制剂(t=1.06,P=0.009)相比更强。hUCMSC-Exos使RA FLSs凋亡率升高[(48.68±0.84)%,t=12.33,P<0.001]。hUCMSC-Exos可降低RA FLSs上清液中TNF-α[(29.6±1.0)pg/ml,t=10.78,P<0.001]、IL-6[(20.1±0.7)pg/ml,t=7.96,P<0.001]、IL-1β[(9.28±0.23)pg/ml,t=6.14,P<0.001]、IL-8[(108.0±3.8)pg/ml,t=1.21,P<0.001]的分泌水平。hUCMSC-Exos可降低RA FLSs中p-NF-κB-p65/NF-κB-p65蛋白表达水平(0.Objective To investigate the effect of hUCMSC-exos on the expression levels of HDAC in different isotypes of RA FLSs,and to elucidate the possible mechanism of hUCMSC-exos on the apoptosis of RA FLSs by regulating HDAC.Methods hUCMSC and hUCMSC-Exos were isolated and identified.RT-qPCR was used to detect the changes in HDAC mRNA expression levels in FLSs after hUCMSC-Exos intervention,and the most affected HDAC types were identified.Western blot was used to detect the levels of FLS HDAC1 protein and the expression levels of NF-κB p65 and phospho-NF-κB p65(Ser 536)in the blank control group,hUCMSC group,hUCMSC-Exos group,Trichostatin A(TSA)group and HDAC1 Inhibitor(Pyroxamide)group.To investigate the effects of hUCMSC-Exos on HDAC expression and NF-κB activity in FLSs.Flow cytometry was used to detect the effect of hUCMSC-Exos on the apoptosis of FLSs.ELISA was used to detect the effects of hUCMSC-Exos on the secretion of TNF-α,IL-6,IL-1βand IL-8 by FLSs.Flow cytometry and ELISA were used to detect the apoptosis level and pro-inflammatory cytokine secretion level of RA FLSs in the blank control group,NF-κB Inhibitor(pyrrolidine dithiocarbamate(PDTC)group,hUCMSC-Exos group and PDTC+hUCMSC-Exos co-intervention group.Whether inhibition of NF-κB affects the regulatory effect of hUCMSC-Exos on RA FLSs was further explored.All experimental data conforming to the normal distribution were compared by one-way ANOVA.LSD-t test was used for pin-group comparison,and independent sample t test was used for two-sample comparison.Results Cultured primary hUCMSC were adherently grown spindle-shaped cells,and hUCMSC-Exos were saucer-shaped membranous vesicles,both of which met the identification criteria.hUCMSC-Exos reduced the expression level of HDCA1 mRNA[(0.932±0.091),t=2.19,P<0.001]and protein[(0.204±0.012),t=8.28,P<0.001]in RA FLSs,and the inhibitory effect was stronger than that of hUCMSC(t=1.09,P=0.009)and HDAC1(t=11.29,P=0.013)Inhibitor.hUCMSC-Exos increased the apoptosis rate of RA FLSs[(48.68±0.84)%,t=12.33,P<0

关 键 词：关节炎 类风湿 滑膜细胞 间充干细胞 外泌体 信号传导 细胞凋亡 

分 类 号：R593.22[医药卫生—内科学]