目的 观察脂肪来源间充质干细胞外泌体(exosomes from adipose derived mensenchymal stem cells,ADSCs-exo)对小鼠树突状表皮T细胞(dendritic epidermal T cells,DETC)及皮肤创面炎症反应调控的影响.方法 自2023年1-12月,解放军总医院第四医学中心烧伤整形医学部采用Ⅰ型胶原酶消化法提取ADSCs,收集ADSCs培养上清液利用差速超速离心法提取ADSCs-exo.原代提取并培养DETC,利用流式技术对细胞纯度进行鉴定;利用刀豆蛋白A(concanavalin A,ConA)构建体外DETC细胞炎症模型,用不同浓度的ADSCs-exo对DETC处理后,采用RT-qPCR法检测细胞中IL-2、IL-17A的mRNA表达.构建C57小鼠背部皮肤全层皮肤缺损创面模型,依次分为空白对照组、PBS注射组、ADSCs-exo注射组,蛋白质免疫印迹法检测各时间段创缘皮肤IL-17A、Akt、P-Akt表达情况;利用流式细胞分析技术观察创缘DETC表达情况.结果 DETC体外炎症模型中,ConA 组 IL-2、IL-17A mRNA 表达量均明显高于 Control 组,ConA+ADSCs-exo 各组 IL-2、IL-17A mRNA 表达量相较于 ConA组不同程度降低.在小鼠创面愈合过程中可以观察到ADSCs-exo组小鼠创缘皮肤IL-17A含量要低于PBS组和对照组,而P-Akt表达量要高于PBS组和对照组.小鼠急性创伤造模后第1天,PBS组和ADSCs-exo组创缘DETC表达程度均明显高于对照组.急性创伤后第3天,PBS组创缘DETC表达量仍明显高于对照组;ADSCs-exo组DETC表达出现下降,基本降至对照组水平.结论 ConA能较好诱导活化DETC分泌表达IL-17A和IL-2,而ADSCs-exo能显著抑制其IL-2、IL-17A的表达.在小鼠创面愈合过程中,ADSCs-exo能抑制创缘皮肤IL-17A的表达、提高Akt蛋白磷酸化水平.急性创伤能提高创面DETC表达;ADSCs-exo对于DETC并无明显的趋化富集作用,但却能在较短时间内抑制DETC的表达,避免创面持续、过度的炎症反应发生.
Effects of exosomes derived from adipose mesenchymal stem cells on the regulation of dendritic epidermal T cells and wound inflammation in mice
Objective To investigate the impact of exosomes derived from adipose tissue mesenchymal stem cells(ADSCs-exo)on dendritic epidermal T cells(DETC)and inflammation in cutaneous wounds of mice.Method From January to December 2023,ADSCs were isolated using the type Ⅰ collagenase digestion method,while ADSCs-exo were obtained through differential ultracentrifugation.DETCs were extracted and cultured in primary culture,with cell purity confirmed by flow cytometry.An inflammation model of DETC cells was established in vitro using cleaved soybean protein A(ConA),and the mRNA expressions of IL-2 and IL-17A in cells were analyzed by RT-qPCR.A full-thickness skin defect wound model was created on the back skin of C57 mice,which were then divided into a blank con-trol group,a PBS injection group,and an ADSCs-exo injection group.The levels of IL-17A,Akt,and P-Akt at the wound edge were as-sessed by Western blotting.The expression of DETCs at the wound edge was observed via flow cytometry.Results In the DETC in vitro inflammation model,the mRNA expressions of IL-2 and IL-17A were significantly elevated in the ConA group compared to the Control group.However,in the ConA+ADSCs-exo groups,there was a noticeable decrease in mRNA expressions of IL-2 and IL-17A when com-pared to the ConA group.During the mouse wound healing process,lower levels of IL-17A content were detected at the skin edge of wounds treated with ADSCs-exo compared to those treated with PBS or left untreated(control).Additionally,higher expression levels of P-Akt were observed at these sites as well.On day one after acute trauma,both PBS and ADSCs-exo treatments resulted in significantly increased migration and enrichment degree of DETC cells at wound edges compared to control samples.By day three post-injury,while DETC expression remained significantly higher for PBS-treated wounds relative to controls;its expression decreased within ADSCs-ex-o-treated wounds and reached similar levels as seen in control samples.Conclusion ConA can stimulate activated DETC to secrete IL-17A and IL-2,whereas ADSCs-exo significantly suppress the expression of IL-2 and IL-17A.During the process of wound healing in mice,ADSCs-exo inhibit the expression of IL-17A while promoting P-Akt expression.The expression of DETC at the wound site can be upregulated in response to acute trauma;although ADSCs-exo do not exhibit obvious chemotactic enrichment effect on DETC,the inhibition of DETC expression effectively impedes the occurrence of persistent and excessive inflammatory reactions in the wound,thereby ensuring a more controlled and regulated healing process.
Adipose derived mensenchymal stem cellsExosomesDendritic epidermal T cellsInflammation
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