间充质干细胞调节巨噬细胞极化在重症急性胰腺炎中的作用及其机制研究

Macrophages release a large number of proinflammatory cytokines and trigger inflammatory cascade reaction, which is the main reason for the rapid development of severe acute pancreatitis (SAP) from local to systemic inflammation. Studis found that mesenchymal stem cells (MSCs) can reduce the release of pro-inflammatory cytokines of SAP, but the exact mechanism is not clear so far. Trauma-related studies found that MSCs played an anti-inflammatory role and inhibited macrophage M1 polarization. Our previous studies showed that SAP macrophages could be induced to undergo M1 polarization in rat; MSCs could down-regulate the expression of Card9 gene in macrophages; disruption of Card9 gene could inhibit NFкB activation, reduced macrophages to M1 polarization. Therefore, we speculated that MSCs play a protective role in SAP by down-regulating the expression of Card9 gene and inhibiting the downstream NFкB activity of macrophages, thereby inhibiting the polarization of macrophages to M1. Through the study of animal, cells, and clinical aspects, this project intends to adopt the method of genetic perturbation, flow cytometry, iTRAQ differential proteomics, signal transduction chips and reverse blocking and other methods to reveal the protective role of MSCs in SAP and its mechanism of regulating macrophage polarization through Card9-NFкB signal axis. The study will provide new ideas and theoretical basis for the clinical treatment of SAP.

巨噬细胞释放大量促炎细胞因子引发炎症级联反应是重症急性胰腺炎（SAP）从局部迅速发展为全身炎症的主要原因。研究发现间充质干细胞（MSCs）可减少SAP促炎细胞因子释放，但机制不明。在创伤等研究中发现MSCs发挥抗炎作用与抑制巨噬细胞M1极化有关。我们前期研究发现：大鼠SAP巨噬细胞可发生M1极化；MSCs可下调巨噬细胞中Card9表达；干扰Card9基因可抑制NFκB活化，减少巨噬细胞向M1极化。因此，我们推测MSCs通过下调巨噬细胞Card9表达并抑制其下游NFκB的活性，从而抑制巨噬细胞M1极化，在SAP中发挥保护性作用。本项目拟采用基因扰动、流式细胞术、iTRAQ差异蛋白组、信号转导芯片和反向阻断等方法，通过细胞、动物、临床层面的研究，揭示MSCs调节巨噬细胞极化在SAP中的作用及其通过Card9-NFкB信号轴调节巨噬细胞极化的机制，为SAP的临床治疗提供新的思路和理论依据。

巨噬细胞释放大量促炎细胞因子引发炎症级联反应是重症急性胰腺炎（SAP）从局部迅速发展为全身炎症的主要原因。研究发现间充质干细胞（MSCs）可减少SAP促炎细胞因子释放，但机制不明。在创伤等研究中发现MSCs发挥抗炎作用与抑制巨噬细胞M1极化有关。我们前期预实验发现：大鼠SAP巨噬细胞可发生M1极化；MSCs可下调巨噬细胞中Card9表达，CARD9、NFκB参与SAP炎症反应。因此，本研究中我们希望证实MSCs对SAP巨噬细胞的极化调节作用及探究其作用机制。本研究建立SAP大鼠模型及体外M1型巨噬细胞极化模型，应用尾静脉注射MSCs、转染腺病毒体内外干扰CARD9表达及建立共培养体系等多种形式，检测巨噬细胞极化类型变化、CARD9表达变化及NFκB磷酸化改变。结果证实MSCs可抑制SAP大鼠腹腔巨噬细胞M1型极化，减轻SAP大鼠炎症反应；MSCs 可下调SAP大鼠 CARD9基因及蛋白表达；干扰SAP大鼠CARD9表达可减少巨噬细胞向M1极化，减少M1型相关细胞因子的产生，减轻SAP大鼠炎症反应；干扰M1型巨噬细胞CARD9表达，上述指标变化与体内实验结果一致，进一步证实CARD9参与巨噬细胞极化。M1型巨噬细胞与MSCs共培养后TNF-α、IL-6mRNA水平及M1型巨噬细胞表面标志物蛋白表达水平均显著降低，提示MSCs可能分泌一些分子引起巨噬细胞向M1极化减少；同时引起CARD9表达水平、NFκB磷酸化水平明显降低，这些结果与干扰M1型巨噬细胞CARD9表达变化一致。同时，体内外实验检测发现MSCs分泌可TSG-6。以上研究从细胞模型、动物模型方面证实SAP早期巨噬细胞趋向于M1型极化。证实MSCs可抑制巨噬细胞向M1型极化，以减轻SAP炎症反应。阐明Card9-NFκB信号轴在MSCs抑制巨噬细胞M1极化中的作用，为SAP的临床治疗提供新的思路和理论依据。

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