PD-L1基因修饰间充质干细胞外泌体诱导VCA免疫耐受的作用及机制研究

Vascularized composite allotransplantation (VCA) is an ideal way for the treatment of limbs and facial deformity, but complications brought by the long-term use of immunosuppressive agents are always difficult problems which need to be solved in clinic. Induction of donor specific immune tolerance is an important research direction in this field. Studies have shown that the mesenchymal stem cells (MSCs)-exosomes have similar biological effects and immune regulatory function with MSCs, and it avoids the defects of stem cell therapy. Programmed cell death ligand 1 (PD-L1) inhibits the activation of T cells in a variety of ways, while MSCs is associated with the expression of programmed death 1 (PD-1) molecule in T cells and has a potential for gene modification. Previous studies have confirmed that MSCs exosomes significantly prolongs the survival time of VCA allografts. This research plans to use gene transfection techniques, prompting MSCs exosomes’ high expression of PD-L1, and make the immune regulation of MSCs exosome itself work, thereby forming local microenvironment of immune suppression, so as to induce T cells being converted to the direction of immune tolerance. The aim is to investigate the possibility and mechanism of induction of donor specific immune tolerance, which would provide an experimental basis for promoting long-term graft survival of clinical VCA.

吻合血管的同种异体复合组织移植（VCA）是治疗肢体残疾和颜面部畸形的理想途径，但如何避免长期服用免疫抑制剂带来的并发症是临床上亟待解决的问题，而诱导供体特异性免疫耐受是该领域的重要研究方向。研究表明，间充质干细胞（MSCs）外泌体具有与MSCs相似的生物学效应和免疫调节功能，且其避免了干细胞治疗的缺陷。程序性细胞死亡-配体1（PD-L1）可通过多种途径抑制T细胞活化，而MSCs外泌体则可促进T细胞表达程序性死亡受体（PD-1)分子水平上调，且其具有基因修饰的潜力。课题组前期研究证实MSCs外泌体能显著延长VCA移植物存活时间。本研究拟采用基因转染技术，促使MSCs外泌体高表达PD-L1，并发挥MSCs外泌体自身的免疫调节作用，形成局部免疫抑制微环境，诱导移植受体T细胞向免疫耐受方向转化，探讨其诱导供体特异性免疫耐受的可能性及机制，为临床VCA移植物长期高质量存活提供实验基础。

吻合血管的同种异体复合组织移植是治疗复合组织缺损最理想的方法，然而宿主抗移植物反应限制了这项技术的发展。免疫抑制剂的应用仍然是目前治疗复合组织移植免疫排斥的最主要措施，但是长期使用免疫抑制剂必然给患者带来严重的并发症。诱导免疫耐受可能是减少或替代免疫抑制药物长期应用的根本性措施。本研究旨在通过外泌体修饰技术来诱导免疫耐受。我们用携带PD-L1的慢病毒感染骨髓间充质干细胞而得到富含PD-L1的外泌体。我们发现，这种修饰后的外泌体其诱导幼稚T细胞向调节性T细胞转化的效率比普通的未修饰的外泌体的效率高，且其有明显的抑制幼稚T细胞增殖的能力。同时，和Treg细胞的产生相关的细胞因子的mRNA得表达也明显上调，如白细胞介素-2和干扰素-γ。在动物实验中，通过静脉注射的PD-L1修饰过的外泌体，移植的大鼠前肢的存活时间明显延长，Treg细胞的比例明显提高，且免疫相关的炎症因子呈现了类似的趋势。通过这些实验，我们证实了PD-L1修饰的外泌体在诱导免疫耐受方面的高效性。