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
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青海大学附属医院,西宁 810000

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R-33

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Effect of microRNA-26a-overexpressed rat epidermal-stem-cell-derived exosomes on wound healing in rats with deep second-degree burns
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Qinghai University Affiliated Hospital, Xining 810000, China

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    摘要:

    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。

    Abstract:

    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, NCGFP-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-EPSCExos on angiogenesis. A rat model of deep second-degree burns was established and separated into control, NC-EPSCExos, 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 7th, 14th and 21st days 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.

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王献珍,李 毅,吴晓伟,崔 强.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[J].中国比较医学杂志,2022,32(11):34~42,56.

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  • 收稿日期:2022-03-22
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  • 在线发布日期: 2023-01-18
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