Establishment of murine model for auto immune hepatitis (AIH)

自身免疫性肝炎（AIH）小鼠模型的建立

• 批准号: 14570523
• 负责人: OKUMURA Akihiko
• 金额: $ 2.24万
• 依托单位: Aichi Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570523/
• 关键词: murine hepatitis model; CYP2D6; CD4^+-T cell; CD8^+-T cell; CD4^+ CD25^+-T cell (T-reg); liver infiltrating lymphocytes; 肝内浸潤リンパ球(LIL); CYP2D6; ベクター; 融合蛋白; CD4+T細胞; CD8+T細胞; 特異的免疫応答; 肝障害; 抗体産生

Background : We established murine hepatitis model by immunizing mice with human P450IID6 (CYP2D6), the target molecule of LKM-1 antibody. In the present study, we investigated the mechanism of liver cell injury in AIH, including the dynamics of T-reg, using our murine hepatitis model. Materials and methods : Eight week old C57BL/6 male mice were immunized with β-gal-CYP2D6 (0.1μmol), the fusion protein of β-gal and CYP2D6, intraperitoneally. Serum levels of ALT and anti-CYP2D6, and histologic changes in the liver were tested weekly until 4 week after immunization. To determine the proportion of CD4^+-T cell, CD8^+-T cell, and T-reg, liver infiltrating lymphocytes (LIL) and splenocytes (SP) were collected, stained with anti-CD4-PE in combination with anti-CD8-FITC or anti-CD25-FITC, and run on FACS flow cytometer, collecting data on 1x10^4 cells, analyzed using Cell Quest software. Results : Serum ALT level reached the peak at 2 week after immunization and returned to normal level at 4 … More week in the mice immunized with β-gal-CYP2D6. Histologically, periportal infiltration of the inflammatory cells and focal necrosis in the lobule was observed in the livers of the β-gal-CYP2D6-immunized mice. When LIL were used for the FACS analysis, the ratio of CD4/CD8 decreased at 1 week after immunization, synchronized with the elevation of ALT levels, then recovered gradually in parallel with the resolution of ALT flare. When SP were used, however, the change of CD4/CD8 was marginal, indicating the different dynamics of CD4^+- and CD8^+-T cells in SP from that in LIL during the course of hepatitis. The frequency of CD4^+ CD25^+-T cell (T-reg) in SP were similar to LIL. The frequency of T-reg in LIL decreased immediately at 1 week after immunization, and gradually increased to the same level as pre-immunization. T-reg in SP decreased more slowly than that in LIL, reached minimum at 2 week after immunization, then increased similarly to T-reg in LIL. Conclusions : Immunization of C57BL/6 mice with β-gal-CYP2D6 resulted in inflammation in the liver. Our results using FACS analysis indicated the predominance of CD8^+T cells in the livers with hepatitis. Further, the frequency of T-reg changed drastically during the flare of ALT levels, implies the possibility that T-reg play some roles on the course of hepatitis. Less

背景：我们用LKM-1抗体的靶分子人P450 IID 6（CYP 2D 6）免疫小鼠，建立小鼠肝炎模型。在本研究中，我们研究了AIH肝细胞损伤的机制，包括T-reg的动态，使用我们的小鼠肝炎模型。材料与方法：用β-gal与CYP 2D 6的融合蛋白β-gal-CYP 2D 6（0.1μmol）腹腔免疫8周龄C57 BL/6雄性小鼠。每周检测血清ALT和抗CYP 2D 6水平以及肝脏组织学变化，直至免疫后4周。为了确定CD 4 ^+-T细胞、CD 8 ^+-T细胞和T-reg的比例，收集肝脏浸润淋巴细胞（LIL）和脾细胞（SP），用抗CD 4-PE联合抗CD 8-FITC或抗CD 25-FITC染色，并在FACS流式细胞仪上运行，收集1 × 10^4个细胞的数据，使用Cell Quest软件进行分析。结果：血清ALT水平在免疫后2周达到高峰，4周恢复正常 ...更多信息 免疫小鼠中的β-gal-CYP 2D 6。组织学上，在β-gal-CYP 2D 6免疫小鼠的肝脏中观察到门静脉周围炎性细胞浸润和小叶中的局灶性坏死。当用LIL进行流式细胞术分析时，CD 4/CD 8比值在免疫后1周下降，与ALT水平的升高同步，然后随着ALT的消退而逐渐恢复。而使用SP时，CD 4/CD 8比值变化不大，表明在肝炎过程中SP和LIL中CD 4 ^+-和CD 8 ^+-T细胞的动态变化不同。SP组CD 4 ^+ CD 25 ^+-T细胞（T-reg）的比例与LIL组相似。LIL中T-reg的频率在免疫后1周立即下降，并逐渐上升至免疫前水平。SP组T-reg的下降速度比LIL组慢，在免疫后2周达到最低，随后又与LIL组T-reg相似地上升。结论：β-gal-CYP 2D 6免疫C57 BL/6小鼠可引起肝脏炎症反应。我们的流式细胞仪分析结果表明，肝炎患者肝脏中CD 8 ^+T细胞占优势。此外，T-reg的频率在ALT水平的爆发期间急剧变化，暗示T-reg在肝炎过程中发挥某些作用的可能性。少