过表达microRNA-26a大鼠表皮干细胞来源外泌体对深Ⅱ度烧伤大鼠创面愈合的影响

Effect of microRNA-26a-overexpressed rat epidermal-stem-cell-derived exosomes on wound healing in rats with deep second-degree burns

目的 探究过表达microRNA-26a(miR-26a)的大鼠表皮干细胞(EPSC)来源外泌体(Exos)对深Ⅱ度烧伤大鼠创面愈合的影响。方法 体外培养大鼠EPSC,用携带miR-26a-绿色荧光蛋白(GFP)的慢病毒颗粒和阴性对照(NC-GFP)转染以上调miR-26a表达,并从未转染的EPSC、NC-GFP转染的EPSC或miR-26a-GFP转染的EPSC中分离Exos,透射电子显微镜(TEM)和纳米粒子追踪分析(NTA)观察Exos形态和大小,蛋白质印迹(Western blot)检测Exos表面标志物CD9、CD63和CD81的表达,实时荧光定量聚合酶链反应(RT-qPCR)检测EPSC及EPSC-Exos中miR-26a表达,CCK-8法检测EPSC的增殖活性;小管形成实验评估miR-26a-EPSC-Exos对血管生成的影响。建立大鼠深Ⅱ度烧伤模型,分为对照组(control组)、NC-EPSC-Exos组和miR-26a-EPSC-Exos组,每组30只。NC-EPSC-Exos组和miR-26a-EPSC-Exos组大鼠给予相应的Exos干预,control组大鼠给予等体积的PBS,每周1次,持续3周。分别在烧伤后第7、14和21天,观察各组大鼠烧伤创面状况,计算创面愈合率;HE染色观察创面组织病理学变化,并进行组织学评分;免疫组织化学法检测创面CD31阳性表达,计算微血管密度(MVD)。结果 miR-26a转染可显著升高EPSC及其Exos中miR-26a表达,促进EPSC细胞增殖(P<0.05);TEM和NTA分析结果显示,Exos呈球形,直径范围在40~150 nm, CD9、CD63和CD81均呈阳性;小管形成实验结果显示,miR-26a-EPSC-Exos可显著增加管长度,促进内皮细胞血管生成(P<0.05)。体内动物实验结果显示,miR-26a-EPSC-Exos可显著加速深Ⅱ度烧伤大鼠创面愈合和修复,增加组织学评分和MVD(P<0.05)。结论 过表达miR-26a的EPSC-Exos可促进深Ⅱ度烧伤大鼠创面愈合。

Objective To investigate the effect of exosomes(Exos) derived from rat epidermal stem cells(EPSC) overexpressing microRNA-26a(miR-26a) on wound healing in rats with deep second-degree burns. Methods Rat EPSCs were cultured in vitro and transfected with lentiviral particles carrying miR-26a-green fluorescent protein(GFP) and negative control(NC-GFP) to up-regulate the expression of miR-26a. Exos were separated from untransfected, NC-GFP-transfected, NC-GFP-transfected and miR-26a-GFP-transfected EPSCs. Transmission electron microscopy and nanoparticle tracking analysis were performed to observe the shape and size of the Exos. Western blot was performed to measure the expression of Exo surface markers CD9, CD63 and CD81. Real-time fluorescent quantitative PCR was performed to measure the expression of miR-26a in EPSC and EPSC-Exos, and the CCK-8 method was applied to measure the proliferation activity of EPSC. A tubule formation experiment was performed to evaluate the effect of miR-26a-EPSC-Exos on angiogenesis. A rat model of deep second-degree burns was established and separated into control, NC-EPSC-Exos, and miR-26a-EPSC-Exos groups, with 30 rats in each group. The NC-EPSC-Exos and miR-26a-EPSC-Exos groups were given corresponding Exos intervention, and the control group was given an equal volume of PBS once a week for 3 weeks. On the 7, 14and 21days after rats were burnt, the burn wound conditions of the rats in each group were observed and the wound healing rate was calculated. HE stainning was performed to observe histopathological changes of the wounds, and histological scores were obtained. An immunohistochemical method was employed to measure the positive expression of CD31 on the wound and calculate the microvessel density. Results miR-26a transfection significantly increased the expression of miR-26a in EPSC and its Exos and promoted the proliferation of EPSCs(P<0.05). Transmission electron microscopy and nanoparticle tracking analysis result showed that Exos were spherical, with a diameter ranging from 40 nm to 150 nm, and positive for CD9, CD63, and CD81. The tubule formation experiments showed that miR-26a-EPSC-Exos was able to significantly increase the tube length and promote endothelial cell angiogenesis(P<0.05). The result of the in vivo experiments showed that miR-26a-EPSC-Exos significantly accelerated wound healing and repair in rats with deep second-degree burns and increased the histological score and microvessel density(P<0.05). Conclusions EPSC-Exos overexpressing miR-26a can promote wound healing in rats with deep second-degree burns.