IL-1β活化脂肪间充质干细胞对小肠缺血再灌注损伤的修复作用及机制研究

Intestinal ischemia-reperfusion (IR) injury is very common in a variety of critical diseases like trauma and shock; promoting the healing of intestinal IR injury will decrease the morbidity and mortality. Pathological features of intestinal IR injury include release of oxygen free radicals, excessive inflammatory reaction, cellular apoptosis and microcirculatory disturbance. Our previous studies indicated that adipose tissue-derived mesenchymal stem cells (ADMSC) suppress inflammatory reaction, improve blood supply and promote gut tissue regeneration, which are realized by upregulation of the expression of COX-2-PGE2 signaling pathway. Activation is the prerequisite for ADMSC to suppress inflammation and promote tissue regeneration, and a few studies have indicated that ADMSC was functional ascribed to its in vivo activation. Our preliminary data suggest that ADMSC significantly upregulate the expression of COX-2-PGE2 signaling with the in vitro stimulation of IL-1β. We hypothesize that activated ADMSC by IL-1β will promote the healing of intestinal IR injury through upregulation of the expression of COX-2-PGE2 signaling. This project will evaluate whether IL-1β-activated ADMSC promote the healing of intestinal IR injury, and we also explore the role of COX-2-PGE2 signaling in IL-1β-activated ADMSC for promotion of the healing of intestinal IR injury.

小肠缺血再灌注(IR)损伤在危重疾病(如创伤和休克)中十分常见；促进小肠IR损伤的愈合显著降低危重疾病并发症的发生率和死亡率。小肠IR损伤的病理生理特征为氧自由释放、炎症反应、细胞凋亡和微循环障碍。我们的前期研究结果提示脂肪间充质干细胞(ADMSC)抑制炎症反应、改善微循环和促进消化道组织再生；其机制为ADMSC通过上调表达COX-2-PGE2信号通路而发挥作用。ADMSC活化是其抑制炎症和促进组织再生的前提；目前，少数研究提示体外活化ADMSC能显著增强其组织修复能力。我们的预实验提示IL-1β体外活化ADMSC能够显著上调COX-2-PGE2信号通路的表达；因此，我们预测IL-1β活化的ADMSC通过上调表达COX-2-PGE2而促进小肠IR损伤的修复。本课题拟通过动物和细胞实验评价IL-1β体外活化ADMSC促进小肠IR损伤修复的作用，并探讨与COX-2-PGE2信号通路的内在关联。

COX-2-PGE2是IL-1β活化间充质干细胞（ADMSCs）重要的信号通路，调控ADMSCs功能。（1）体外研究中发现，使用不同浓度的IL-1β活化ADMSCs后，ADMSCs细胞中COX-2和PGE2表达显著升高。通过特异性干扰COX-2基因表达，IL-1β对ADMSCs细胞的活化功能显著减弱。（2）小肠缺血再灌注损伤（IR）的动物实验中发现，IL-1β活化的ADMSCs较ADMSCs显著减轻IR损伤，减少小肠上皮细胞凋亡和炎性反应。通过表达谱测序发现，与IR对照组相比，IL-1β活化的ADMSCs组有900多个基因表达水平发生改变；且通过Real-time PCR技术进一步证实了凋亡相关基因(Apaf1 and S100a9)和免疫功能相关基因（CXCL11, CXCL2, Tnfrsf22, CXCR2 and CXCR4)表达水平较IR组显著下调。通过GO生物信息学分析，IL-1β活化的ADMSCs组调控的基因主要涉及炎性反应、粘附分子、防御反应等生物过程；通过KEGG信息分析，发现IL-1β活化的ADMSCs组显著抑制TNF、PPAR、PI3K-Akt 和NF-κb信号通路的活化。Western blot进一步证实磷酸化P65、ERK 和Akt蛋白水平显著降低，进一步证实RNA测序结果。（3）慢病毒特异性干扰ADMSCs中COX-2表达显著降低IL-1β活化的ADMSCs对IR损伤的修复作用，提示COX-2在IL-1β对ADMSCs的活化和IL-1β活化的ADMSCs对IR损伤的修复中起关键性作用。此课题深入研究了IL-1β活化的ADMSCs促进小肠缺血再灌注损伤的修复作用，探讨了COX-2-PGE2信号通路在IL-1β活化ADMSCs中的作用，为ADMSCs的临床转化应用提供了证据。本课题资助发表论文2篇，待发论文2篇（1篇北大核心和1篇SCI论文），培养硕士研究生2名。本课题直接经费18万，支出8.68万，各项支出基本与预算相符。剩余经费9.32万元，剩余经费计划用于本项目研究后续支出。