脂肪源性间充质干细胞外泌体在大鼠全层皮肤缺损创面愈合中的机制研究  

Mechanism research of adipose-derived mesenchymal stem cell-derived exosomes in wound healing of full-thickness skin defect in rats

作　　者：付文 王向臣[1] 王延桂[1] 孙绍韶 FU Wen;WANG Xiangchen;WANG Yangui;SUN Shaoshao(Department of Clinical Laboratory,Yan-taishan Hospital,Yantai 264003,China)

机构地区：[1]烟台市烟台山医院检验科,山东省烟台市264003

出　　处：《组织工程与重建外科》2023年第4期342-351,379,共11页Journal of Tissue Engineering and Reconstructive Surgery

摘　　要：目的探究脂肪源性间充质干细胞外泌体(hADSC-Exo)在大鼠全层皮肤缺损创面愈合中的作用机制。方法分离并鉴定hADSC与hADSC-Exo。将人真皮微血管内皮细胞(hMECs)分为对照组、200μg/L重组人表皮生长因子处理的阳性对照组与100μg/mL hADSC-Exo处理的hADSC-Exo组,通过CCK-8、划痕实验、血管形成实验,检测hMECs增殖、迁移与血管形成能力。60只大鼠随机分为对照组、阳性对照组、hADSC组与hADSC-Exo组,每组15只。所有大鼠于背部制备全层皮肤缺损创面,每组分别于大鼠尾静脉注射200μL 1×PBS、100μg/kg重组人表皮生长因子、200μL约含1×10^(6)个hADSC的1×PBS、200μL含200μg hADSC-Exo的1×PBS。每组随机保留3只大鼠,分别于0、3、7、10、12 d拍摄创面图像并计算创面面积。每组其余大鼠随机分为4组,每组3只,分别于3、7、10、12 d采集未愈合创面组织并制成石蜡切片,HE、Masson染色观察创面愈合情况,免疫组化检测炎症与创面愈合相关蛋白表达情况。结果细胞实验结果显示,与对照组相比,阳性对照组、hADSC-Exo组细胞增殖活性、细胞迁移率升高(P<0.05),管腔长度与分支点数量增加(P<0.05);与阳性对照组相比,hADSC-Exo组细胞增殖活性、细胞迁移率进一步升高(P<0.05),管腔长度与分支点数量进一步增加(P<0.05)。动物试验结果显示,与对照组相比,阳性对照组、hADSC组与hADSC-Exo组皮肤创面愈合率、CD206、Ⅰ型胶原蛋白(CollagenⅠ)、CollagenⅢ、水通道蛋白3(AQP3)表达水平升高(P<0.05),肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-6、CD68、角蛋白1(KRT1)表达水平与CollagenⅠ/CollagenⅢ比例下降(P<0.05),新生表皮组织更为完整,厚度增加,表皮下可见成纤维细胞增殖,创面新生胶原沉积明显增加;与阳性对照组相比,hADSC组与hADSC-Exo组CD206、CollagenⅠ、CollagenⅢ、AQP3表达水平进一步升高(P<0.05),TNF-α、IL-6、CD68、KRT1表达水平进一步下降(P<0.05)Objective To explore the mechanism of adipose-derived mesenchymal stem cell-derived exosomes(hADSC-Exo)on wound healing of full-thickness skin defect in rats.Methods hADSC and hADSC-Exo were isolated and identified.Human microvascular endothelial cells(hMECs)were divided into control group,positive control group(treated with 200μg/L recombinant human epidermal growth factor)and hADSC-Exo group(treated with 100μg/mL hADSC-Exo).The proliferation,migration and angiogenesis of hMECs were detected by CCK-8,scratch and angiogenesis experiments.A total of 60 rats were randomly divided into control group,positive control group,hADSC group and hADSC-Exo group,15 rats in each group.Full-layer skin defect wounds were prepared on the back of all rats.Each group was injected with 200μL 1×PBS,100μg/kg recombinant human epidermal growth factor,200μL 1×PBS containing 1×10^(6) hADSC and 200μL 1×PBS containing 200μg hADSC-Exo,re-spectively.Three rats were randomly reserved in each group.The wound images were taken and the wound area was calculated on the 0,3,7,10 and 12 days respectively.The rest of the rats in each group were randomly divided into 4 groups,with 3 rats in each group.The unhealed wound tissues were collected on the 3,7,10 and 12 days respectively,and tissue samples were prepared into paraffin sections.HE and Masson staining were used to observe the wound healing.The expression of proteins related to inflammation and wound healing was detected by immunohistochemistry.Results The results of cell experiment showed that compared with the control group,the cell proliferation activity,cell migration rate,lumen length and the number of branching points increased in the positive control group and hADSC-Exo group(P<0.05).Compared with the positive control group,the cell proliferation activity,cell migration rate,lumen length and the number of branching points in hADSC-Exo group were further increased(P<0.05).The results of animal test showed that compared with the control group,the wound healing rate and the expr

关 键 词：脂肪源性间充质干细胞 外泌体 全层皮肤缺损 创面愈合 

分 类 号：R628[医药卫生—整形外科]