气压调控感染结核巨噬细胞极化的机制及其对T细胞的影响研究

Negative pressure wound therapy can cure tuberculosis wound including the bone and joint tuberculosis with wound, and change the status of the Th1/Th2.Macrophage polarization reflects and adjusts the immune response of Th1/Th2.Therefore, there is an important relationship between the negative pressure and the polarization of macrophages.However, the specific mode of action remains to be elucidated. In this study, the transcriptome sequencing showed that A20 was highly expressed in the wound tissue, Macrophages infected with MTB by negative pressure cuture highly express M1 polarization related gene and NF-KB gene.It was also found that the expression of A20 was up-regulated in the patients with tuberculosis wound and the expression of A20 was down regulated in macrophages infected with MTB. But under these two conditions, the negative pressure can reduce the expression of A20 and polarization to M1.A20 and NF-kB were negatively correlated. Presumably the pressure may control of macrophages polarization infected with MTB through the A20-NF-kB.This study intends to study the expression of A20 related signal axis and the polarization characteristics of macrophages through the cell model, animal model, clinical specimens, to study the regulatory mechanism and the Effect on T cells by applying positive and negative pressure, gene over expression and inhibition test and cell co-culture system. Provide the basis for finding new anti-tuberculosis methods.

负压创面疗法可治愈结核创面患者（含骨关节结核创面），改变Th1/Th2状态；巨噬细胞M1/M2极化反映并调节Th1/Th2免疫应答，提示负压与巨噬细胞极化存在重要联系，但具体作用方式尚待阐明。本课题组高通量测序发现A20（TNFAIP3）在结核创面组织高表达，感染MTB的巨噬细胞经负压培养后M1极化相关基因高表达且NF-kB上调。还发现结核创面患者A20表达上调，相反感染MTB的巨噬细胞A20表达下调，但负压均能使这两种情况中的A20下调并向M1极化，且A20、NF-kB呈负相关。据此推测气压可能通过A20-NF-KB调控感染MTB的巨噬细胞极化。本项目拟通过细胞模型、动物模型、临床标本研究A20相关信号轴表达及巨噬细胞极化特性，通过施加正负气压、调节基因表达及细胞共培养深入研究其调控机制及对T细胞的影响，为寻找抗结核新方法提供依据。

项目的背景：骨关节结核是最常见的肺外结核，包括脊柱结核和关节结核，病变累及皮下皮肤会形成创面，属于深层结核创面，发病率高治疗难度大。负压创面疗法可治愈结核创面患者（含骨关节结核创面），改变Th1/Th2状态；巨噬细胞M1/M2极化反映并调节Th1/Th2免疫应答，提示负压与巨噬细胞极化存在重要联系，但具体作用方式尚待阐明。.主要研究内容：.1.基因和蛋白筛查.1.1生物信息学与生物检测筛查结核感染后各mRNA和蛋白含量变化。.1.2构建相关ceRNA调控网络.2.调控机制研究.2.1气压通过XIST-miR125b-5p-A20调节感染结核后巨噬细胞极化功能；.2.2气压通过A20-NF-KB调节感染结核后巨噬细胞极化功能；.3.MTB感染的巨噬细胞经气压干预后，对T细胞功能的影响。.重要结果、关键数据：.1、分析3个基因表达综合公共数据库中显示结核感染后共有91个基因表达明显异常。.2、MTB感染后，miR-125b-5p在细胞和小鼠中表达下调，负压处理后miR-125b-5p表达恢复；在MTB感染的细胞和小鼠中，XIST表达上调，并可被负压逆转；XIST通过与miR-125b-5p结合来抑制miR-125b-5p的表达；XIST和miR-125b-5p的表达依赖于ESAT-6并调节MTB的存活；MTB感染后miR-125b-5p下调通过A20阻断NF-κB通路促进巨噬细胞极化为M2表型；MTB感染后，负压处理通过XIST/miR-125b-5p/A20轴使巨噬细胞向M1表型分化。.3、H37Rv细菌感染RAW264.7细胞负压模型构建成功；H37Rv细菌感染RAW264.7细胞并施加负压后，可以促进RAW264.7细胞向M1极化，并且NF-κB mRNA表达量明显上升；A20成功沉默后可以对NF-κB表达起到调控作用；A20过表达后NF-κB蛋白会发生表达变化，可能会影响到巨噬细胞的极化；A20过表达后会影响想过信号通路，影响 TNF-α、IL-1βmRNA和蛋白表达变化；.4、感染MTB的M2巨噬细胞经过负压治疗后使CD4+T细胞免疫应答向Th1方向改变。.科学意义：.本研究深入研究其机制，可以为结核治疗策略改进和方法更新提供新思路。