Establishment of murine model for auto immune hepatitis (AIH)

背景：用LKM-1抗体的靶分子人P450IID6(CYP2D6)免疫小鼠，建立小鼠肝炎模型。在本研究中，我们利用我们的小鼠肝炎模型，研究了AIH中肝细胞损伤的机制，包括T-reg的动态变化。材料和方法：8周龄C57BL/6雄性小鼠用β-GAL-CYP2D6融合蛋白(0.1mmol.1Mol)免疫C57BL/6雄性小鼠。μ-GAL融合蛋白为β-GAL-CYP2D6。每周检测血清ALT、抗-CYP2D6水平及肝脏组织学改变，直至免疫后4周。收集肝脏浸润性淋巴细胞(LIL)和脾细胞(SP)，用抗CD8-PE和抗CD8-FITC或抗CD25-FITC联合染色，FACS流式细胞仪检测1×10~(-4)细胞数，用Cell Quest软件进行统计分析。结果：丙氨酸氨基转移酶在免疫后2周达高峰，4…时恢复正常β-GAL-CYP2D6免疫组小鼠的免疫时间延长。组织学观察，β-Gal-CYP2D6免疫组小鼠肝脏门脉周围炎性细胞浸润，小叶局灶性坏死灶。用LIL进行FACS分析，免疫后1周，CD4/CD8比值下降，与ALT升高同步，随后随着ALT的消退而逐渐恢复。而应用SP后，CD_4/CD_8比值变化不大，提示在肝炎病程中SP和LIL中CD_4~+~+、CD_8~+~+T细胞的动态变化不同。SP患者外周血中CD4~+CD25~+T细胞(T-reg)的比例与LIL相似。免疫后1周，LIL中T-reg频率即下降，并逐渐回升至免疫前水平。免疫后2周，SP组T-reg下降速度较LIL慢，2周时降至最低，而后升高，与LIL相似。结论：β-Gal-CYP2D6免疫C57BL/6小鼠可引起肝脏炎症反应。流式细胞仪分析结果表明，肝炎患者肝组织中CD8~+T细胞占优势。此外，T-reg的频率在ALT水平的升高过程中发生了剧烈的变化，提示T-reg可能在肝炎的发病过程中起一定的作用。较少

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