PK34修饰间充质干细胞在结核病治疗中的作用及分子机制研究

At present, poor tissue repairmen, the side effects and drug resistance of anti-tuberculosis drugs have become a serious problem in the treatment of tuberculosis. It is of great significance to develop a method with high efficiency and little damage to eliminate Mycobacterium tuberculosis (MTB) infection and a method to repair of the lesion. Mesenchymal stem cells (MSCs) can be differentiated into many kinds of cells, repair tissues, and have immunomodulatory function. MSCs can be applied into tissue injury repairing and targeted therapy for MTB infection. However, MSCs is now only a remedy for anti-tuberculosis therapy. MSCs can inhibit the immune activity of T cells, and assist in the immune escape and long-term latency of MTB. It will leave a hidden danger for the recurrence of tuberculosis. Compared with traditional anti-tubercular agents, antimicrobial peptides have low toxic side effects and are not easy to produce drug resistance. They have broad antimicrobial spectrum, eliminate MTB rapidly, function specifically, and become new tools for clinical treatment of tuberculosis. It is urgent to elucidate whether antimicrobial peptide combined with MSCs is beneficial to the treatment of tuberculosis and the effect and mechanism of this combination. In this project, we will first construct the antimicrobial peptide expressing MSCs, then culture these MSCs with MTB infected macrophages and infusion these MSCs to MTB infected mice. We will observe the antimicrobial effects and tissue repairments, in vitro and in vivo, elucidate the anti-tuberculosis molecular mechanism of antimicrobial peptide modified MSCs, provide a theoretical basis for the use of antimicrobial peptide modified MSCs in the treatment of tuberculosis, and provide a new treatment option for refractory tuberculosis.

目前结核病治疗面临抗结核药物的毒副作用和耐药性、组织修复性差等问题，开发新的抗结核分枝杆菌（Mycobacterium tuberculosis，MTB）感染和病灶修复方法具有重要意义。间充质干细胞（mesenchymal stem cells，MSCs）能修复组织，具免疫调节功能，可用于MTB的靶向治疗与组织损伤修复。但MSCs现在仅作为MTB的补救治疗，且MSCs可抑制T细胞活性，协助MTB的免疫逃逸，为复发留下隐患。抗菌肽相比抗结核药，毒副作用低、不易产生耐药性、抗菌谱广、杀菌快速、作用专一。抗菌肽联合MSCs是否有利于抗结核治疗，及两者联合抗结核病的作用及其机制亟待阐明。本项目将表达抗菌肽的MSCs与感染巨噬细胞共培养、输注治疗感染小鼠，观察抗菌效果及组织修复情况，阐明其抗结核感染的分子机制，为使用抗菌肽修饰MSCs治疗结核病提供理论依据，为难治结核病提供新的治疗途径。

自该项目立项实施以来，项目负责人积极组织项目组研究人员对该项目进行了研究，按计划开展实验。项目组分离并培养了小鼠密质骨MSCs（cbMSCs），流式鉴定结果显示cbMSCs表面标志物CD105、CD29、Sca-1和CD45阳性率分别为31.8%、95.5%、96.8%和1.4%，符合文献对小鼠MSCs表面标志物的相关报道，分离培养成功；cbMSCs成骨和成脂分化结果显示，镜下可见矿化结节和胞内脂滴，说明细胞具有多向分化能力；以来自结核分枝杆菌噬菌体D29的抗菌肽PK34为基础，构建了PK34基因过表达慢病毒载体，构建与包装成功完成，滴度为109 IU/ml；使用该慢病毒成功感染了cbMSCs，流式细胞仪测定GFP荧光报告阳性率为74.5%；转染后的细胞经成骨和成脂分化结果显示具有多向分化能力。为后续用于人体治疗考虑，项目组同时分离并培养了人脐带MSCs（ucMSCs），流式鉴定结果显示ucMSCs高表达表面标志物CD73、CD90、CD105和CD44，阳性率分别为98.4%、99.9%、99.6%和99.8%，不表达CD45/CD34/CD11b/CD19/HLA-DR，共阳率为0.0%，分离培养成功；ucMSCs成骨和成脂分化结果显示，镜下可见矿化结节和胞内脂滴，说明细胞具有多向分化能力；使用PK34基因过表达慢病毒成功感染了ucMSCs，流式细胞仪测定GFP荧光报告阳性率为87.7%；转染后的细胞经成骨和成脂分化结果显示具有多向分化能力。项目组为开展项目需要，使用化学合成法制备了合成PK34，同时构建了PK34原核表达体系，并表达纯化了重组PK34；药物敏感试验结果显示，重组PK34较合成PK34抗菌活力更高，对BCG、结核分枝杆菌 H37 Ra均具有较好的抑菌活性。为PK34的后续实验研究和应用，项目组还制备了重组PK34的单抗隆抗体，抗体效价> 256,000，S/C.O = 1.15。项目组还制备了带有信号肽和PK34基因的穿梭质粒GV141和GV219，后续将检测在其他真核细胞中表达PK34，并观察其与合成PK34、原核细胞表达PK34的区别，期望可以拓宽这一抗菌肽的应用领域。综上所述，该项目取得了较好的进展，但还需要后续进一步进行深入研究，项目组期待实验结果能够对结核分枝杆菌感染的辅助治疗提供有利的理论依据和更为广阔的应用前景。