脂肪来源干细胞胞外囊泡调控转化生长因子β-Smad信号通路抑制瘢痕增生的分子机制研究  

Study on molecular mechanism of extracellular vesicles derived from adipose-derived stem cells regulating TGF-β-Smad signaling pathway to inhibit scar hyperplasia

作　　者：朱元正[1] 易阳艳[1] 王江文 聂佳莹 王朝慧[1] 吴舒[1] 杨娟敏 Zhu Yuanzheng;Yi Yangyan;Wang Jiangwen;Nie Jiaying;Wang Zhaohui;Wu Shu;Yang Juanmin(Department of Plastic Surgery,the Second Affiliated Hospital of Nanchang University,Nanchang 330006,China)

机构地区：[1]南昌大学第二附属医院整形美容科,330006

出　　处：《中华整形外科杂志》2020年第10期1114-1120,共7页Chinese Journal of Plastic Surgery

基　　金：国家自然科学基金地区项目(81660326);江西省自然科学基金重点项目(20171ACB20037);江西省自然科学基金面上项目(20192BAB205056)。

摘　　要：目的探讨脂肪干细胞-细胞外囊泡(ASC-EVs)对肌成纤维细胞转分化过程中转化生长因子β(TGF-β)-Smad信号通路的调控作用。方法脂肪干细胞来源于南昌大学第二附属医院整形美容科行吸脂术患者的抽吸脂肪。真皮成纤维细胞来源于同一科室包皮环切术患者自愿捐赠的皮肤组织。将脂肪抽吸物离心纯化后经酶消化提取脂肪来源于细胞(ASCs),扩增后行流式细胞术检测表面蛋白标记物。收集第3~5代ASCs上清液提取胞外囊泡(EVs),透射电镜观察微观形态,纳米颗粒跟踪分析仪NanoSight检测粒径分布,流式细胞术检测其膜表面标志性蛋白CD63、Alix和TSG101。用PKH67荧光标记的ASC-EVs与真皮成纤维细胞共培养后,共聚焦显微镜观察细胞对EVs的摄取情况。通过TGF-β1持续诱导真皮成纤维细胞5 d,并添加50、100μg/ml浓度的ASC-EVs,通过免疫荧光染色,RT-PCR和蛋白质印迹法检测α-平滑肌肌动蛋白(α-SMA)以及Smad-2/3/4的表达。结果流式细胞术结果提示,第3代ASCs表面标记物CD73、CD49d、CD90、CD105为阳性,CD34、CD45为阴性。透射电镜下ASC-EVs为圆形膜性囊泡,边缘清晰,由双层磷脂膜包裹。95%以上的ASC-EVs粒径为(30.0~261.0)nm,平均(166.0±86.1)nm。EVs高表达标志蛋白CD63、Alix和TSG101。共聚焦显微镜下可见携带绿色荧光的ASC-EVs被真皮成纤维细胞摄取并分布在胞浆内,部分ASC-EVs在细胞核周围分布。α-SMA免疫荧光染色后,经连续5 d的TGF-β1诱导,共聚焦显微镜下见真皮成纤维细胞中α-SMA绿色荧光的表达显著提高,而采用50μg/ml和100μg/ml ASC-EVs作用的真皮成纤维细胞中,α-SMA的表达较未经ASC-EVs作用的真皮成纤维细胞显著降低,但不呈现浓度依赖;细胞α-SMA和Smad-2/3/4的mRNA转录水平较未经ASC-EVs作用的真皮成纤维细胞显著下降,α-SMA和Smad-2/3/4的蛋白表达也显著降低。此外,ASC-EVs可显著降低细胞分泌至上清液中的Ⅰ型胶原的含量,Objective This study aims to explore the potential effects of adipose-derived stem cell-extracellular vesicles(ASC-EVs)on TGFβ-Smad signaling pathway during myofibroblast trans-differentiation in vitro.Methods ASCs were isolated from liposuction and flow cytometry was used to detect the surface protein markers.ASC-EVs were isolated from the supernatant of the third to fifth generation ASCs,and the microscopic morphology was observed by transmission electron microscope.The particle size distribution was detected by nano-particle tracking analyzer NanoSight and the membrane surface marker proteins CD63,Alix and TSG101 were detected by flow cytometry.The uptake of EVs by dermal fibroblasts co-cultured with PKH67 fluorescence labeled ASC-EVs was observed by confocal microscope.Dermal fibroblasts were continuously induced by TGFβ1 for five days,and ASC-EVs at the dose of 50 and 100μg/ml were added.The expression ofα-SMA and Smad-2/3/4 were detected by immunofluorescence staining,RT-PCR and Western Blot.Results The results of flow cytometry showed that the surface markers CD73,CD49d,CD90 and CD105 of the third generation ASCs,were positive,and CD34 and CD45 were negative.Under transmission electron microscope,ASC-EVs was a round membranous vesicle with clear edge and surrounded by bilayer phospholipid membrane.The particle size of more than 95%of the ASC-EVs was distributed between 30 nm to 261 nm,with an average of(166.0±86.1)nm.The specific marker proteins of extracellular vesicle,CD63,Alix and TSG101 were highly expressed.Under confocal microscope,ASC-EVs with green fluorescence were uptake by dermal fibroblasts and distributed in the cytoplasm,and part of ASC-EVs was distributed around the nucleus.TGF-β1 induced a significant increase in the expression ofα-SMA in dermal fibroblasts,and the addition of 50 or 100μg/ml ASC-EVs reduced the expression ofα-SMA genes and proteins,but did not show a dose-dependent manner.The gene and protein expression changes of Smad-2/3/4 were consistent withα-SMA.In addition,A

关 键 词：瘢痕 转化生长因子Β 脂肪干细胞 细胞外囊泡 肌成纤维细胞 

分 类 号：R622[医药卫生—整形外科]