The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice
The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice
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DOI：
10.3791/3644
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发表时间：
2012-02-01
影响因子：
1.2
通讯作者：
Christen, Urs
中科院分区：
文献类型：
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作者：
Hintermann, Edith;Ehser, Janine;Christen, Urs
自身免疫性肝炎是一种罕见但危及生命的肝脏自身免疫性疾病，病因不明（1，2）。在过去，已经进行了许多尝试来产生反映人类疾病特征的动物模型（3-5）。然而，在各种模型中，疾病的诱导相当复杂，并且肝炎通常只是短暂的（3-5）。因此，我们开发了一种简单的小鼠模型，该模型使用2型自身免疫性肝炎（AIH-2）中的主要人类自身抗原，即hCYP 2D 6作为触发剂（6）。识别hCYP 2D 6的1型肝肾微粒体抗体（LKM-1）抗体是AIH-2的标志（7，8）。通过腺病毒构建体（Ad-2D 6）将hCYP 2D 6递送到野生型FVB或C57 BL/6小鼠中，所述腺病毒构建体确保触发抗原直接递送到肝脏。因此，随后的局部炎症为随后的自身免疫发展产生了肥沃的土壤（9）。静脉和腹腔注射Ad-2D 6的组合是诱导对肝脏的持久自身免疫性损伤的最有效途径（第1节）。在这里，我们提供了一个详细的协议，如何在CYP 2D 6模型中诱导自身免疫性肝病，以及如何评估肝损伤的不同方面。首先，通过眶后采血测定指示肝细胞破坏的标志物（如转氨酶）的血清水平以及hCYP 2D 6抗体的滴度（第2节）。其次，hCYP 2D 6特异性T细胞应答的特征在于从脾和肝收集淋巴细胞。为了获得纯的肝脏淋巴细胞，通过门静脉用PBS灌注肝脏（第3节），在胶原蛋白中消化并在Percoll梯度上纯化（第4节）。通过用hCYP 2D 6肽刺激并通过流式细胞术鉴定IFN γ产生细胞来分析hCYP 2D 6特异性T细胞的频率（第5节）。第三，通过肝切片的免疫组织化学确定细胞浸润和纤维化（第6节）。这种分析方案必须在疾病开始后多次进行，以证明模型的慢性性质。必须评估以hCYP 2D 6特异性T细胞和/或B细胞的频率和活性为特征的免疫应答的强度以及肝损伤和纤维化的程度，以用于随后评价预防、延迟或消除肝的自我破坏过程的可能治疗。
Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology(1,2). In the past many attempts have been made to generate an animal model that reflects the characteristics of the human disease(3-5). However, in various models the induction of disease was rather complex and often hepatitis was only transient(3-5). Therefore, we have developed a straightforward mouse model that uses the major human autoantigen in type 2 autoimmune hepatitis (AIH-2), namely hCYP2D6, as a trigger(6). Type 1 liver-kidney microsomal antibodies (LKM-1) antibodies recognizing hCYP2D6 are the hallmark of AIH-2(7,8). Delivery of hCYP2D6 into wildtype FVB or C57BL/6 mice was by an Adenovirus construct (Ad-2D6) that ensures a direct delivery of the triggering antigen to the liver. Thus, the ensuing local inflammation generates a fertile field(9) for the subsequent development of autoimmunity. A combination of intravenous and intraperitoneal injection of Ad-2D6 is the most effective route to induce a long-lasting autoimmune damage to the liver (section 1). Here we provide a detailed protocol on how autoimmune liver disease is induced in the CYP2D6 model and how the different aspects of liver damage can be assessed. First, the serum levels of markers indicating hepatocyte destruction, such as aminotransferases, as well as the titers of hCYP2D6 antibodies are determined by sampling blood retroorbitaly (section 2). Second, the hCYP2D6-specific T cell response is characterized by collecting lymphocytes from the spleen and the liver. In order to obtain pure liver lymphocytes, the livers are perfused by PBS via the portal vein (section 3), digested in collagen and purified over a Percoll gradient (section 4). The frequency of hCYP2D6-specific T cells is analyzed by stimulation with hCYP2D6 peptides and identification of IFN gamma-producing cells by flow cytometry (section 5). Third, cellular infiltration and fibrosis is determined by immunohistochemistry of liver sections (section 6). Such analysis regimen has to be conducted at several times after initiation of the disease in order to prove the chronic nature of the model. The magnitude of the immune response characterized by the frequency and activity of hCYP2D6-specific T and/or B cells and the degree of the liver damage and fibrosis have to be assessed for a subsequent evaluation of possible treatments to prevent, delay or abrogate the autodestructive process of the liver.