脂肪间充质干细胞外泌体通过miR-145抑制小鼠增生性瘢痕血管生成及纤维增生  

Adipose-derived mesenchymal stem cell exosomes inhibit hypertrophic scar angiogenesis and fibrogenesis in mice via miR-145

作　　者：阿丽米热·伊力哈木[1] 卡米力江·买买提明[2] 美尔瓦提[3] 李朝阳[1] Alimire·Yilihamu;Kamilijiang·Maimaitiming;Meierwati;LI Zhao-yang(Department of Plastic Surgery,People’s Hospital of Xinjiang Uygur Autonomous Region,Urumqi Xinjiang 830001,China;Department of Maxillofacial Surgery,People’s Hospital of Xinjiang Uygur Autonomous Region,Urumqi Xinjiang 830001,China;Department of Clinical Psychology,People’s Hospital of Xinjiang Uygur Autonomous Region,Urumqi Xinjiang 830001,China)

机构地区：[1]新疆维吾尔自治区人民医院整形外科,新疆乌鲁木齐830001 [2]新疆维吾尔自治区人民医院颌面外科,新疆乌鲁木齐830001 [3]新疆维吾尔自治区人民医院临床心理科,新疆乌鲁木齐830001

出　　处：《局解手术学杂志》2022年第10期870-877,共8页Journal of Regional Anatomy and Operative Surgery

基　　金：新疆维吾尔自治区自然科学基金(2021D01C112)。

摘　　要：目的探究miR-145在脂肪间充质干细胞(ADSCs)来源外泌体(Exo)抑制小鼠增生性瘢痕(HS)血管生成与纤维增生中的作用。方法从人皮下脂肪组织分离培养ADSCs,经成脂或成骨分化诱导培养基诱导后,采用油红O染色或茜素红染色检测细胞成脂、成骨分化能力,流式细胞术测定ADSCs表面标志物CD29、CD34、CD45、CD90和CD105表达;使用含miR-145过表达的慢病毒感染ADSCs后提取Exo,透射电镜观察颗粒物的形态,纳米颗粒跟踪分析仪分析粒径大小,Western blot检测Exo表面标志性蛋白Alix、Tsg101和CD81表达,实时荧光定量PCR检测miR-145的相对表达量。将40只SPF级健康雄性C57/BL6小鼠按照随机数字表法分为对照组、模型组、Exo组和miR-145-Exo组,每组10只,除对照组外,其余3组小鼠均构建HS模型,Exo组和miR-145-Exo组小鼠分别在背部伤口处皮下注射100μL Exo与转染miR-145的Exo,对照组和模型组注射等体积PBS;14 d后处死各组小鼠并切取瘢痕组织,HE染色和Masson染色观察皮肤组织病理学变化及胶原沉积情况,免疫组化染色测定血管内皮生长因子(VEGF)表达,Western blot检测α-平滑肌肌动蛋白(α-SMA)、转化生长因子-β1(TGF-β1)、Ⅰ型胶原蛋白(Collagen Ⅰ)及Ⅲ型胶原蛋白(Collagen Ⅲ)的蛋白表达水平。结果分离的细胞经茜素红染色和油红O染色后,可见细胞内沉积钙盐,有明显脂滴形成,CD105、CD90、CD29呈阳性表达,而CD34、CD45呈阴性表达,说明成功分离获得ADSCs;透射电镜观察到颗粒物为球形囊泡,粒径峰值约为128 nm,Exo标志性蛋白Alix、Tsg101和CD81均呈阳性表达,说明成功分离到Exo,且经miR-145慢病毒转染的ADSCs来源Exo中miR-145相对表达水平升高(P<0.05)。相较于模型组,Exo组和miR-145-Exo组的小鼠皮肤组织损伤减轻,HS区域减少,且真皮层变薄,胶原沉积减少,组织纤维化得到缓解,VEGF阳性表达率降低,α-SMA、TGF-β1、Collagen Ⅰ及Collagen Ⅲ的蛋白表达水平�Objective To investigate the role of miR-145 in adipose-derived mesenchymal stem cells(ADSCs)-derived exosomes(Exo)in inhibiting the angiogenesis and fibrogenesis of hypertrophic scar(HS)in mice.Methods ADSCs were isolated and cultured from human subcutaneous adipose tissue.After induction with adipogenic or osteogenic differentiation induction medium,oil red O staining or alizarin red staining were used to detect the adipogenic and osteogenic differentiation abilities of the cells,and flow cytometry was used to determine the expression of ADSCs surface markers CD29,CD34,CD45,CD90 and CD105.ADSCs were infected with miR-145-overexpressed lentivirus,and then the Exo was extracted.The morphology of particles was observed by transmission electron microscope,the particle size was analyzed by nanoparticle tracking analyzer,the expression of Exo surface marker proteins Alix,Tsg101 and CD81 were detected by Western blot,and the relative expression of miR-145 was detected by real-time fluorescence quantitative PCR.Forty SPF healthy male C57/BL6mice were divided into the control group,the model group,the Exo group and the miR-145-Exo group according to the random number table method,with 10 mice in each group.Except the control group,the other three groups of mice were all constructed with HS models.Mice in the Exo group and the miR-145-Exo group were subcutaneously injected with 100μL of Exo and miR-145-transfected Exo at the back wound,respectively;mice in the control group and the model group were injected with equal volumes of PBS.After 14 days,the mice in each group were sacrificed and the scar tissue was excised,HE staining and Masson staining were used to observe histopathological changes and collagen deposition of skin,immunohistochemical staining was used to determine the expression of vascular endothelial growth factor(VEGF),Western blot was used to detect the protein expression levels of α-smooth muscle actin(α-SMA),transforming growth factor-β1(TGF-β1),typeⅠcollagen(CollagenⅠ)and type Ⅲ collagen(Coll

关 键 词：增生性瘢痕 MIR-145 脂肪间充质干细胞 外泌体 血管生成 纤维化 

分 类 号：R622[医药卫生—整形外科]