PD-1抑制剂相关性肝炎小鼠模型的构建方法

Establishment of a Mouse Model of PD-1 Inhibitor-Associated Hepatitis

摘要: 目的：免疫检查点抑制剂诱发的肝炎是肿瘤免疫治疗中常见的免疫相关不良反应。本研究旨在通过比较不同造模条件，探讨利用PD-1单抗结合T细胞有丝分裂原ConA建立小鼠PD-1抑制剂相关性肝炎模型的方法。方法：小鼠分为正常组、ConA组和ConA + PD-1单抗联用组。ConA组及联用组小鼠尾静脉注射ConA (20 mg/kg)；联用组于ConA注射后6小时或24小时腹腔注射PD-1抑制剂(250 μg/只)，正常组与ConA组同期注射等量生理盐水。分别于ConA注射后12、24、48、72 h结束实验。收集血清、肝脏。采用生化试剂盒检测血清丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)水平，苏木精–伊红(H&E)染色观察肝组织病理学改变，免疫组化法检测肝脏中T细胞浸润情况以及干扰素-γ (IFN-γ)的分泌水平。结果：ConA联合PD-1单抗在不同检测时间点均可不同程度地加重ConA诱导的炎症反应。其中，ConA注射6小时后给予PD-1单抗干预6小时(即ConA注射后12 h)所致的炎症反应最为显著：与ConA组相比，联用组小鼠血清ALT和AST水平显著升高。H&E染色结果显示，联用组小鼠肝细胞坏死增多，肝组织炎症浸润程度较ConA组加重。免疫组化结果显示，联用组小鼠肝组织中CD3⁺ T细胞浸润显著增加，IFN-γ的表达水平亦较ConA组明显升高。结论：本研究通过PD-1单抗联合ConA成功建立了PD-1抑制剂相关性肝炎小鼠模型。在ConA注射后6小时再给予PD-1单抗干预6小时，可显著加重ConA诱导的免疫性肝炎背景下的肝损伤，其机制可能与促进肝脏T细胞(CD3⁺ T细胞)浸润及上调IFN-γ表达有关。

Abstract: Objective: Immune checkpoint inhibitor-induced hepatitis is a common immune-related adverse event in cancer immunotherapy. This study aimed to establish a mouse model of PD-1 inhibitor-associated hepatitis by combining a PD-1 monoclonal antibody with the T-cell mitogen Concanavalin A (ConA) and comparing different modeling conditions. Methods: Mice were divided into a normal control group, a ConA group, and a ConA + anti-PD-1 combination group. Mice in the ConA and combination groups received an intravenous tail vein injection of ConA (20 mg/kg). The combination group received an intraperitoneal injection of anti-PD-1 antibody (250 μg/mouse) at either 6 hours or 24 hours post-ConA injection. The normal and ConA groups received an equivalent volume of saline at the same time points. Experiments were terminated at 12, 24, 48, and 72 hours after ConA injection. Serum and liver samples were collected. Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using commercial kits. Liver histopathology was evaluated by hematoxylin and eosin (H&E) staining. T-cell infiltration and the expression levels of tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) were assessed by immunohistochemistry. Results: The combination of ConA and anti-PD-1 exacerbated ConA-induced inflammation to varying degrees at all tested time points. The most pronounced inflammatory response was observed when the anti-PD-1 antibody was administered 6 hours after ConA injection, followed by assessment 6 hours later (i.e., 12 hours post-ConA). In this group, serum ALT and AST levels were significantly elevated compared to the ConA-only group. H&E staining revealed increased hepatocyte necrosis and more severe inflammatory infiltration in the combination group. Immunohistochemistry showed a significant increase in CD3⁺ T-cell infiltration, as well as elevated expression of IFN-γ in the liver and spleen tissues of the combination group compared to the ConA group. Conclusion: This study successfully established a mouse model of PD-1 inhibitor-associated hepatitis by combining a PD-1 monoclonal antibody with ConA. Administering the anti-PD-1 antibody 6 hours after ConA injection for a 6-hour intervention period significantly exacerbated liver injury in the context of ConA-induced immune-mediated hepatitis. This mechanism may be related to the promotion of hepatic T-cell (CD3⁺ T cell) infiltration and the upregulation of IFN-γ expression.