调节性T细胞在药物性急性肝脏免疫损伤中的效应机制及其特性研究

Acetaminophen(APAP, analgesic and antipyretic drug)overdose leads to acute liver injury and even death.The investigation by our team and other goups demonstrate that damage-associated molecule patterns released from APAP-induced necrotic hepatocytes activate immune response to aggravate liver injury,which is described as two-hit model.Up to now, the pathogenic immunological mechanisms have been extensively studied.However, the immune homeostasis and recovery during APAP-induced liver injury have not been well elucidated. Moreover,the effective strategy for immunological intervention of drug-induced liver injury has not be constructed.In the current study,we focus on the role of regulatory T cells(Treg cells) in APAP-induced liver injury and the effect of hepatic inflammatory microenviroment on the property of Treg cells. The percentages and numbers of Treg cells in the liver and spleen will be measured after acetaminophen challenge.Treg cell depletion and adoptive tranfer will be performed to establish the function of Treg cells. Molecular lineage analysis,antibody blocking and cytokine overexpression in the liver will be conducted to study the functional mechanism of Treg cells.The effect of hepatic inflammatory microenvrionment on the phenotypes,function and trafficking of regulatory T cells will be investigated with flow cytometry,in vitro cytokine stimulation and in vivo receptor blocking. This research will povide evidence that Treg cells can be used in the prevention and treatment of drug-induced liver injury.

解热镇痛药对乙酰氨基酚过量可导致急性肝脏损伤，严重时危及生命。近期国际同行和我们的研究均发现对乙酰氨基酚导致肝细胞死亡只是肝脏损伤的始发原因，坏死肝细胞释放的"损伤相关模式分子"所激发的免疫损伤（二次打击）是主要原因。由于目前主要集中研究病理性免疫损伤机制，对其中的免疫自稳和恢复机制研究甚少，还不能提出有效的干预对策。本课题将研究调节性T细胞在对乙酰氨基酚诱导肝脏损伤中的作用及肝脏炎性微环境对其影响。首先观察此病理过程中是调节性T细胞在肝脏、脾脏中的数量变化；通过细胞清除和过继转输实验，确证调节性T细胞在此病理过程中所起的作用；通过分子谱系分析、抗体阻断、肝脏过表达等实验，探讨调节性T细胞发挥作用的分子机制；通过细胞表型和功能分子流式检测、体外刺激及体内受体阻断实验，观察肝脏炎性微环境对调节性T细胞表型、功能和迁移的影响。为了解和利用调节性T细胞预防和治疗药物性肝脏损伤提供新线索。

对乙酰氨基酚过量导致的急性肝脏损伤已危害到公众健康。毒性中间代谢产物是导致肝细胞死亡的首要因素，而死亡的肝细胞释放的DAMP分子激活的免疫应答则会造成肝脏的二次损伤，但目前对其中免疫介导的肝脏损伤机制还未研究清楚。本研究旨在探索免疫细胞及其互作在对乙酰氨基酚诱导肝脏损伤中的作用及作用机制。我们给予小鼠400mg/kg的对乙酰氨基酚，24小时后即可诱导出严重的肝脏损伤，血清ALT水平升高，肝脏中出现大量坏死区域，同时肝组织中和血清中的IFN-γ和TNF-α水平均升高。通过基因缺陷小鼠及抗体中和实验确定IFN-γ介导了APAP诱导的肝脏损伤。流式细胞分析、细胞清除实验、缺陷小鼠实验确定IFN-γ和TNF-α主要来源于活化的CD4+T细胞。同时，在对乙酰氨基酚诱导肝脏损伤的过程中，调节性T细胞在肝脏和脾脏中的比例和数量都增加。细胞清除及过继转输实验证实调节性T细胞可有效减轻对乙酰氨基酚诱导的肝脏损伤。抗体中和及细胞因子过表达实验证实调节性T细胞发挥保护性作用依赖于抑制性细胞因子IL-10和TGF-β。肝脏炎症微环境中IFN-γ诱导的趋化因子CXCL9和CXCL10显著增加，且肝脏中CXCR3阳性的调节性T细胞占总的调节性T细胞比例高于脾脏。趋化因子过表达实验证实CXCL10可招募外周的调节性T细胞进入肝脏发挥保护作用。因此，我们确定免疫应答在药物性肝脏损伤中发挥重要作用，且免疫细胞之间存在负反馈调控的互作机制，调控免疫反应有助于治疗药物性肝脏损伤。