复方苦参汤协同干细胞微囊泡运载PD-L1治疗溃疡性结肠炎的机制研究

Ulcerative colitis（UC） is a refractory intestinal non-specific inflammatory disease. Unbalanced intestinal immune homeostasis caused by excessive activation of T cells is one of the important pathogenesis of UC. Studies have shown that blocking the binding of PD-1 to PD-L1 can deactivate the T cell's braking system and initiate immune response. Our previous study found that the expression of PD-1 on the surface of UC intestinal T cells was increased and positively correlated with the degree of intestinal inflammation. This may have a compensatory effect on reducing intestinal inflammation. Therefore, we hypothesized that promoting the binding of PD-1 to PD-L1 in UC inhibited T cell activation, induced Treg production, and established intestinal mucosal tolerance to alleviate intestinal inflammation. Based on the previous research basis, we found that stem cell-derived MVs avoid many stem cell cancer transformation and low transplantation efficiency, while retaining many repair functions such as anti-oxidation, anti-inflammatory and anti-apoptosis of stem cells. It is a good carrier for membrane protein receptor expression. Therefore, this study intends to use stem cell MVs as a carrier, and use the filter extrusion method and lentivirus technology to obtain stem cell MVs overexpressing PD-L1, and promote the colonization of MVs into the intestine by the synergistic action of compound Sophorae Decoction, mediating immune tolerance through PD- L1/PD-1, maintaining immunosuppression, restoring intestinal mucosal barrier, which can further improves the efficacy of treatment of UC, and provides strategies and ideas for exploring new treatments.

UC是一种难治性的肠道非特异性炎症性疾病，T细胞过度活化导致的肠道免疫稳态失衡是其重要发病机制。研究表明阻断PD-1与PD-L1结合可以解除T细胞刹车系统，启动免疫应答。我们前期研究发现UC肠道T细胞表面PD-1表达增加且与炎症程度呈正相关，可能对减轻肠道炎症具有代偿作用，因此我们假设：在UC中促进PD-1与PD-L1结合可以抑制T细胞活化，诱导Treg生成，建立肠黏膜耐受以缓解肠道炎症。结合前期研究基础，我们发现干细胞来源MVs在保留干细胞抗氧化、抗炎、抗凋亡等诸多修复功能的基础上，避免了干细胞癌性转化，移植效率不高等问题，是转运膜蛋白受体的良好载体。因此本课题拟将MVs作为载体，利用滤器挤压法和慢病毒技术得到过表达PD-L1的干细胞MVs，在复方苦参汤的协同作用下促进MVs向肠道定植，通过PD-L1/PD-1介导免疫耐受，恢复肠道免疫稳态，进一步提高UC的疗效，为探索新治法提供思路。

背景：UC是一种难治性的肠道非特异性炎症性疾病，T细胞过度活化导致的肠道免疫稳态失衡是其重要发病机制。通过促进 PD-1 与 PD-L1 结合，可能达到诱导 Treg 的生成，抑制 Th17 淋巴细胞活化，建立肠粘膜耐受以缓解肠道炎症的功能。干细胞来源MVs在保留干细胞抗氧化、抗炎、抗凋亡等诸多修复功能的基础上，避免了干细胞癌性转化，移植效率不高 等问题，是转运膜蛋白受体的良好载体。复方苦参汤以清热祛湿为主要治法的中药复方，具有很好的临床疗效。 .主要研究内容：我们首先通过骨髓贴壁培养法获得间充质干细胞，然后通过慢病毒转染技术以及低温超速离心法的方法将 PD-L1 加载至干细胞 MVs 的膜表面，通过WB鉴定PD-L1在MVs上高表达。接下来将PD-L1-MVs, si-PD-L1-MVs通过尾静脉注射至大鼠体内，在复方苦参汤的协同作用下，研究尾静脉注射过表达 PD-L1 的 MVs 对 UC的免疫调节作用以及复方苦参汤的协同作用，提高治疗 UC的疗效。.重要结果及关键数据：免疫组化实验发现，与Control组相比，DSS组BACL-2、BAX、NF-kB、TNF-α蛋白表达明显升高；与DSS组相比，其他治疗组BACL-2、BAX、NF-kB、TNF-α蛋白的表达在治疗干预后，则不同程度地降低。PD-L1-EVs+CSD降低最明显，PD-L1-EVs次之，SiPD-L1-EVs介于PD-L1-EVs与Mesalazine组之间。Elisa发现与Control组比较，DSS组结肠组织促炎因子IFN-γ、IL-2、IL-17的浓度显著升高，抑炎因子TGF-β浓度显著降低；其中PD-L1-EVs+CSD促炎因子表达最低，抑炎因子表达最高，PD-L1-EVs次之。流式检测结果显示， Control组中，脾脏及肠系膜淋巴结Treg细胞比例最高；Th17含量则很少。与Control组比较，DSS组脾脏和肠系膜淋巴结Th17细胞比例显著升高，Treg细胞比例显著降低；与DSS组相比较，各治疗组脾脏和肠系膜淋巴结Treg细胞比例显著升高，Th17细胞比例显著降低。其中， PD-L1-EVs+CSD组脾脏和肠系膜淋巴结Treg细胞比例明显高于DSS组，趋近于Control组。.科学意义：为探索有效的 UC 治疗策略和药物开发提供理论依据和分子靶标。