调节性T细胞来源外泌体对成纤维细胞增殖影响的体外实验研究

Effect of regulatory T cell-derived exosomes on proliferation of fibroblasts in vitro

目的 探讨调节性T细胞(Tregs)来源外泌体对成纤维细胞(CFs)的影响。方法 利用免疫调节性T细胞专用磁珠和差速贴壁离心法分别获得Tregs和CFs,并通过超速离心法与外泌体纯化试剂盒从Tregs上清液中制备外泌体。建立Tregs来源外泌体与CFs共培养模型,并分为Tregs组、CFs组、Tregs来源外泌体与CFs组(Tregs-exo+CFs组)、3倍Tregs来源外泌体与CFs组(3Tregs-exo+CFs组)和CFs来源外泌体与Tregs组(Tregs+CFs-exo组),CCK-8法检测共培养体系CFs增殖变化,免疫荧光法检测CFs对Tregs来源外泌体摄取能力,ELISA法检测共培养体系细胞因子转化生长因子-β(TGF-β)、白介素10(IL-10)水平。结果 Tregs来源外泌体为直径约115 nm的圆形或椭圆形囊泡。与CFs组相比,Tregs来源的外泌体明显促进CFs增殖,且3倍Tregs来源的外泌体显著促进CFs增殖(P<0.01)。免疫荧光证实CFs有效摄取Tregs来源的外泌体,主要集中于细胞质。与CFs组相比,Tregs-exo+CFs组TGF-β、IL-10水平明显升高,3Tregs-exo+CFs组TGF-β、IL-10水平亦显著升高(P<0.01),且TGF-β升高水平超过IL-10升高的水平。结论 体外共培养条件下,调节性T细胞来源外泌体明显促进成纤维细胞的增殖,升高TGF-β、IL-10水平。

Objective To investigate the effect of exosomes derived from regulatory T cells(Tregs)on fibroblasts(CFs).Methods The Tregs and CFs were obtained by special magnetic beads for regulatory T cellsand differential attachment centrifugation,respectively.And exosomes were prepared from Tregs supernatants with using differential centrifugation on Sepharose columns.The co-culture model of Tregs-derived exosomes and CFs were prepared and randomly divided into three groups:Tregs group,CFs group,Tregs-derived exosomes and CFs group,namely:Tregs-exo+CFs group,three fold Tregs-derived exosomes and CFs group,namely:3Tregs-exo+CFs group,CFs-derived exosomes and Tregs group,namely:Tregs+CFs-exo group.The CCK-8 test was used to determine the proliferation of CFs.The uptake of Tregs-derived exosome by CFs was detected by immunofluorescence assay.The level of cytokine transforming growth factor-β(TGF-β)and interleukin 10(IL-10)in the co-culture system was detected by ELISA.Results Tregs-derived exosomes were round or oval vesicles with diameter about 115 nm.Compared with CFs group,Tregs-exo+CFs group significantly promoted CFs proliferation,and 3Tregs-exo+CFs group significantly promoted CFs proliferation(P<0.01).CFs were effectivly uptake of Tregs-exosomes was confirmed by fluorescence confocal microscopy,which was mainly concentrated in the cytoplasm.Compared with CFs group,TGF-βand IL-10 levels in Tregs-exo+CFs group and 3Tregs-exo+CFs group were significantly increased(P<0.01).The increase content level of TGF-βwas significantly higher.Conclusion Under in vitro co-culture conditions,regulatory T-cell-derived exosomes significantly promoted the proliferation of fibroblasts and increased the content levels of TGF-β and IL-10.