脂肪干细胞来源的外泌体对局限性硬皮病成纤维细胞生物学功能的影响  

In vitro study of the effect of adipose stem cell-derived exosomes on the biological function of localizedsclerodermafibroblasts

作　　者：王立铨 黄久佐[1] 俞楠泽[1] 马旭达 李天浩 龙笑[1] Wang Liquan;Huang Jiuzuo;Yu Nanze;Ma Xuda;Li Tianhao;Long Xiao(Department of Plastic Surgery,Peking Union Medical College Hospital,Chinese Academy of Medical Sciences&Peking Union Medical College,Beijing 100032,China)

机构地区：[1]中国医学科学院北京协和医学院北京协和医院整形外科,北京100032

出　　处：《中华整形外科杂志》2023年第6期655-662,共8页Chinese Journal of Plastic Surgery

基　　金：科技部战略性国际科技创新合作重点专项(2020YFE0201600);中国医学科学院医学与健康科技创新工程“十四五”项目(2021-I2M-1-003);北京协和医院中央高水平医院临床科研专项(2022-PUMCH-C-025,2022-PUMCH-A-210)。

摘　　要：目的:探索来源于健康人脂肪干细胞(ADSC)的外泌体对局限性硬皮病成纤维细胞(LSFs)纤维化的体外调控作用。方法:取2021年1月至2022年1月中国医学科学院北京协和医院整形外科10例健康人吸脂手术剩余脂肪组织,体外分离培养ADSC,以超速离心法收集其外泌体(ADSC-Exo);取同期15例局限性硬皮病患者患处皮肤组织,体外分离培养成纤维细胞。采用不同方向的诱导分化染色、纳米粒子追踪分析、透射电子显微镜、PKH26染色和蛋白质印迹法对ADSC及ADSC-Exo进行鉴定。通过细胞外囊泡分泌抑制实验检验ADSC是否通过其外泌体影响LSFs纤维化标志物[Ⅰ型胶原、Ⅲ型胶原、α-平滑肌肌动蛋白(α-SMA)]的表达。取被ADSC-Exo干预的LSFs,通过CCK-8法、划痕实验检测LSFs的增殖和迁移能力。采用实时荧光定量PCR、免疫荧光染色和蛋白质印迹法检测Ⅰ型胶原、Ⅲ型胶原、α-SMA、转化生长因子β(TGF-β)和p-Smad2/3的表达情况。两组比较采用独立样本t检验;多组比较采用方差分析,组间两两比较采用SNK-q检验。结果:成功于体外分离并培养ADSC和LSFs,并提取ADSC-Exo,且证明ADSC-Exo可以内化到LSFs中发挥作用。细胞外囊泡分泌抑制实验证明,ADSC可通过分泌细胞外囊泡降低LSFs的纤维化标志物表达;CCK-8法、划痕实验结果显示,ADSC-Exo干预后LSFs的增殖和迁移能力降低;实时荧光定量PCR、免疫荧光染色和蛋白质印迹结果显示,相较于对照组,ADSC-Exo干预组的Ⅰ型胶原、α-SMA、TGF-β和p-Smad 2/3表达显著降低。结论:在体外,ADSC-Exo可以通过抑制TGF-β/Smad通路来影响LSFs的生物学功能和降低纤维化标志物的表达。Objective To explore the regulatory effect of exosomes derived from healthy human adipose stem cells(ADSC)on the fibrosis of localized scleroderma fibroblasts(LSFs)in vitro.MethodsFrom January 2021 to January 2022,fat from 10 healthy donors in Department of Plastic Surgery,Peking Union Medical College Hospital,Chinese Academy of Medical Sciences was collected by liposuction.Adipose stem cells were isolated and cultured in vitro,and exosomes(ADSC-Exo)were collected.Fibroblasts were isolated from skin tissue of 15 patients with localized scleroderma during the same period and cultured in vitro.Induced differentiation and staining,nanoparticle tracking analysis,transmission electron microscopy,PKH26 staining and Western blotting were used to identify ADSC and their exosomes.The effect of ADSC on the expression of fibrosis markers[collagenⅠ,collagenⅢ,α-smooth muscle actin(α-SMA)]in LSFs through its exosomes was examined by extracellular vesicle secretion inhibition assay.The proliferation and migration abilities of LSFs treated with ADSC-Exo were tested by CCK-8 method and scratch test.Real-time quantitative PCR,immunofluorescence staining and Western blotting were used to detect the expression levels of collagenⅠ,collagenⅢ,α-SMA,transforming growth factorβ(TGF-β)and p-Smad2/3 in LSFs.Independent sample t-test was used to compare between the two groups.One-way ANOVA was used for multi-group comparison,and SNK-q test was used for pairwise comparison.ResultsADSC and LSFs were successfully isolated and cultured in vitro,and ADSC-Exo was extracted.Extracellular vesicle secretion inhibition assay demonstrated that ADSC decreased fibrotic markers of LSFs by secreting extracellular vesicles.Results of CCK-8 and scratch test showed that the proliferation and migration ability of LSFs was decreased by ADSC-Exo treatment.The results of real-time quantitative PCR,immunofluorescence staining and Western blotting showed that compared with the control group,the expressions of collagenⅠ,α-SMA,TGF-βand p-Smad 2/3 in

关 键 词：硬皮病 局限性 纤维化 脂肪干细胞 外泌体 

分 类 号：R593.25[医药卫生—内科学]