重构CX3CR1+调节T细胞调控Th17/Treg平衡治疗原发性胆汁性肝硬化研究

Primary biliary cirrhosis (PBC) is the most common chronic progressive autoimmune liver disease, eventually leading to cirrhosis. The etiology is unknown, which may be associated with breakdown of self tolerance to the pyruvate dehydrogenase complex E2 subunit (PDC-E2) and induce the bile duct epithelial cells injure. Th17 cells are considered to induce autoimmunity injure, and regulatory T cells (Tregs) are crucial for maintaining self-tolerance and down-tuning immune response. Our investigation found that patients with PBC had a relative reduction in the number of Treg cells in peripheral blood, but Th17 cells were found to be increased compared with that in normal control. With the recover of the condition, the number of Tregs were increase. It is proposed that the balance between Th17 and Tregs in both number and function may play an important role in the pathogenesis and development of PBC. Targeted tissue-specific antigen Tregs should shift Th17-Tregs balance for treatment PBC. To verify this hypothesis, the reconstructed CX3CR1+ Tregs generated by the specificity of PDC-E2 163-176 antigen peptides, which targeted identify bile duct cells. The antigen-specific CX3CR1+ Tregs should intervention in Th17-Tregs imbalance, inhibition of the small bile duct inflammation and maintain immune tolerance, then treatment of the PBC by adoptive immunotherapy. The new perspective is expected to explain pathogenesis of PBC and unravel novel strategies to treat PBC.

原发性胆汁性肝硬化（PBC）是最常见慢性进展性自身免疫肝病，最终导致肝硬化。病因不明，可能与自身抗原线粒体主要表位丙酮酸脱氢酶复合物E2亚单位（PDC-E2）免疫耐受缺失，致胆管上皮细胞自身免疫损伤有关。Th17诱导自身免疫发生，而调节性T细胞（Treg）有免疫抑制、维持免疫耐受作用。本课题组发现PBC患者外周血Treg数量下降，Th17表达增加，随着患者病情恢复Treg数量回升，Th17/Treg数量和功能失衡导致PDC-E2耐受缺失与PBC发病密切相关。以组织特异性抗原为靶向的Treg扭转Th17/Treg失衡可能对PBC有治疗作用。为验证此假说，以组织特异性PDC-E2 163-176位抗原肽为靶向、重构识别胆管细胞的CX3CR1+Treg，靶向调控Th17/Treg失衡，抑制小胆管周围炎症，维持免疫耐受，过继免疫治疗PBC。全新角度阐释PBC发病机制，开拓过继免疫及基因治疗新途径。

原发性胆汁性肝硬化（primary biliary cirrhosis, PBC）是以血清抗线粒体抗体（anti-mitochondrial antibodies, AMAs）阳性，肝脏汇管区淋巴细胞浸润，胆管上皮细胞特异性损伤，慢性胆汁淤积为主要特征的自身免疫性疾病。病因不明，可能与自身抗原线粒体表位丙酮酸脱氢酶复合物E2亚单位（pyruvate dehydrogenase complex E2 subunit, PDC-E2）免疫耐受缺失，胆管上皮免疫损伤有关。体液免疫、细胞免疫、胆管上皮细胞凋亡及环境因素在PBC发病进展中起到重要作用。Th17 诱导自身免疫损伤，而调节性T细胞（Treg）有免疫抑制、维持免疫耐受作用。PBC患者外周血Th17 数量升高，Treg 数量降低，Th17/Treg 比例失衡与PBC 发病相关。TGF-β信号通路异常导致外周免疫耐受障碍并引起PBC临床表现；TGF-β信号通路异常导致Foxp3表达异常，而Treg分泌TGF-β、IL-10 等发挥免疫负调控作用。本研究初步结果发现PDC-E2 163-176 位抗原特异性T细胞对胆管上皮细胞有杀伤作用；以人工合成的PDC-E2类似物2OA结合BSA免疫C57BL/6J(B6)小鼠，制造PBC模型，预先或每周给以自然杀伤细胞（NK细胞）抑制剂抗Asialo GM1抗体剔除NK细胞，以研究NK细胞在PBC发生发展中的作用。初步结果发现预先剔除NK细胞，PBC模型小鼠肝脏门脉周围炎症无明显改善，而持续剔除NK细胞后PBC模型小鼠肝脏中小胆管周围炎症减轻，提示NK细胞可能参与破坏PBC免疫耐受，损伤胆小管，从而维持、促进PBC发生、发展。