人脐带MSC来源exosome介导miR-181c调控严重烧伤过度炎症反应的机制研究

Excessive inflammation caused by severe burns often leads to multiple organ dysfunction syndrome, sepsis, and even result in death. Mesenchymal stem cell-derived exosomes (MSC-exosome)play important roles in reduce inflammation in several diseases. We have previously found that ：①human umbilical cord MSC(hUCMSC)-exosome reduced macrophage inflammation, and decresed level of TLR4、NF-κB/P65. ②miR181c is contained in hUCMSC-exosome which significantly decreased in sever burn mice. hUCMSC-exosome and miR181c involved in severe burns excessive inflammation has not been reported before. TLR4 act as a potential targets of miR181c, but there is no report about hUCMSC-exosome and miR-181c in TLR4 signal pathway. Our study proposed by cell model, animal model and clinical samples to investigate hUCMSC-exosome mediated miR181c decreased TLR4 protein expression in severe burns excessive inflammation. Our project will open a nevol avenue for molecular mechanism of hUCMSC-exosome in excessive inflammatory, and provide a new candidate marker for diagnosis and therapeutic target for severe burn excessive inflammation patients.

严重烧伤引起的过度炎症反应常导致多器官功能障碍、脓毒症，甚至患者死亡。研究表明 MSC-exosome可在多种疾病中发挥抗炎作用。我们前期研究发现①人脐带MSC-exosome可降低巨噬细胞内TLR4、NF-κB/P65等炎症相关蛋白表达、减轻巨噬细胞炎症活化；②人脐带MSC-exosome内含有miR-181c，且miR-181c在烧伤后大鼠体内表达明显降低。TLR4是miR-181c的潜在作用靶点，人脐带MSC-exosome是否通过miR-181c抑制TLR4炎症信号通路发挥抗炎作用目前尚无报道。本课题拟从分子、细胞及动物水平研究人脐带MSCs-exosome介导miR-181c下调TLR4蛋白表达在严重烧伤过度炎症反应中的作用和分子机制，为人脐带MSCs-exosome调控严重烧伤过度炎症反应提供一种新思路，也为烧伤患者的临床诊治、预后判断提供新的生物学靶点。

背景：烧伤是仅次于交通事故的第二大意外伤害，严重烧伤后早期发生的过度炎症反应，可导致机体内部平衡严重紊乱，引起多器官功能衰竭，甚至脓毒症的发生，是目前导致严重烧伤患者死亡的主要原因之一。.主要研究内容：本课题主要从人脐带间充质干细胞来源exosome入手，探讨人脐带间充质干细胞来源exosome介导miRNA在调控严重烧伤后过度炎症反应的作用及机制。首先，在体外大量扩增人脐带间充质干细胞，提取细胞上清分离exosome进行数量及质地鉴定，并对exosome内RNA及miRNA组分进行分析；其次，通过建立30%严重烧伤过度炎症反应大鼠模型，检测大鼠血中白细胞、炎症因子，创面炎症细胞、炎症通路相关蛋白及RNA表达水平，并比较hUCMSC-exosome、hSFC-exosome等不同处理对其影响；再次，建立LPS预处理巨噬细胞模型，在体外验证hUCMSC-exosome、hSFC-exosome等不同处理对炎症因子及炎症相关通路蛋白表达变化的影响；最后，在大鼠模型和细胞模型中比较miR-181c高表达hUCMSC-exosome、hUCMSC-exosome、hSFC-exosome及siTLR4等不同处理对炎症细胞浸润、炎症因子分泌及炎症通路相关蛋白表达的影响。.重要结果：研究表明hUCMSC-exosomes来源稳定，产量丰富，且含有大量miRNAs成份。与假伤组大鼠相比，严重烧伤大鼠血中白细胞数量上升，血清促炎因子TNF-α、IL-1β水平上升，抗炎因子IL-10水平下降；创面组织中TLR4、NF-κB/P65、P-P65蛋白表达上升，miR-181c含量下降。.关键数据：给予hUCMSC-exosomes后，可使严重烧伤大鼠白细胞数量下降，促炎因子TNF-α、IL-1β水平下降，抗炎因子IL-10水平上升；创面内TLR4、NF-κB/P65、P-P65蛋白表达明显下降。与hUCMSC-exosomes相比，miR-181c高表达exosomes抑制严重烧伤大鼠创面炎症细胞浸润，减轻炎症因子TNF-α、IL-1β和炎症蛋白TLR4的效果更强，从而下调了NF-κB/P65、P-P65蛋白水平而减少其核转移，并上调抗炎因子IL-10水平，最终抑制严重烧伤后过度炎症反应的发生。.科学意义：本研究揭示hUCMSC-exosome参与严重烧伤后过度炎症反应调控的具体分子机制