ESTABLISHMENT OF AUTOIMMUNE HEPATITIS MODEL BY USING FUSION CELL OF HEPATOCYTE AND DENDRITIC CELL.

肝细胞与树突状细胞融合细胞建立自身免疫性肝炎模型。

• 批准号: 11670536
• 负责人: ZENIYA Mikio
• 金额: $ 2.18万
• 依托单位: Jikei University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670536/
• 关键词: AUTOIMMUNE HEPATITIS; ANIMAL MODEL; DENDRITIC CELL; 肝内T細胞浸潤; HLA-DR4; CD25; AIH; DR4; 樹状細胞; 肝癌細胞; 融合細胞; 実験的自己免疫性肝炎; 細胞障害性T細胞

Backaround: We tried to establish a newly animal autoimmune hepatitis (AIH) model by using the fusion cell of hepatocyte and dendritic cell (DC) to investigate the immimopathogenesis of AIH.Methoth: DC were generated by 5-days culture of bone marrow cells of C57BL/6 female mice with IL-4 and GM-CSF and fusion with well differentiated hepatoma cell Hepal-6 (H-2Db) were carried out in the presence of 50% PEG. Fusion cells were injected to C57BL/6 mice twice a month subcutaneously followed by peritoneal injection of IL-12 (500ng/body) three times a week. Splenocytes were extracted after final IL-12 injection and cultured with IL-2 (50 μg/ml) for 4 days. cytotoxicity of splenocytes against Hepal-6 was analyzed using 4hr 51Cr release assay. As a control, splenocytes obtamed from non treated, IL-12 treated, fusion cells treated mice were studied.Results: Splenocytes extracted from fusion cell treated mice showed Hepa 1-6 specific cytotoxicity (51% cytolysis, E/T ratio80: 1), but not against … More NK cell sensitive Yac-1 cell. T cell population, which were separated using mierobeades and MACS magnetic separation system, of splenocytes obtained from fusion cell treated mice showed Hepa 1-6 specific cytotoxicity (41% cytolysis, EIT ratio=80: 1). But non T cell population did not show cytotoxicity Interestingly, splenocytes extracted from fusion cell and IL-12 treated mice did not show Hepa 1-6 specific cytotoxicity. Splenocytes extracted from non-treated mice nor IL- 12 treated mice didn t show cytotoxicity either. Immuno-histochemical analysis revealed that intrahepatic T cell infiltration was observed in fusion cell and IL-12 treated mice, but not in fusion cell treated mice.Conclusion: These findings indicate that immunization with fusion cells of dendritic cell and Hepa 1-6 may evoke cytotoxic T cell which recoginize the epitope that is shared between Hepa 1-6 and hepatocyte in vivo, but it is not enough to induce hepatocyte injury. IL-12 may participate in the mechanisms of intrahepatic CTL infiltration and hepatocyte injury in this model. Less

背景：我们试图利用肝细胞与树突状细胞(DC)的融合细胞建立一种新的动物自身免疫性肝炎(AIH)模型，以探讨AIH的免疫发病机制。方法：将C57BL/6雌性小鼠的骨髓细胞与IL-4和GM-CSF混合培养5天，并在50%的聚乙二醇液中与高分化的肝癌细胞Hepal-6(H-2DB)进行融合。C57BL/6小鼠每月皮下注射融合细胞两次，然后每周三次腹腔注射IL-12(500 ng/只)。末次注射IL-12后提取脾细胞，加入IL-2(50μg/ml)培养4d。采用4hr-51Cr释放法检测脾细胞对Hepal-6的细胞毒作用。结果：融合细胞处理组小鼠脾细胞具有HEPA1-6特异性细胞毒作用(51%的细胞杀伤率，E/T比为80：1)，但对…无明显的杀伤作用对NK细胞更敏感的Yac-1细胞。融合细胞处理的小鼠脾细胞经MIROBEADS和MACS磁分离系统分离得到的T细胞群具有HEPA 1-6特异性细胞毒作用(41%的细胞杀伤率，EIT比=80：1)。但有趣的是，从融合细胞和IL-12处理的小鼠中提取的脾细胞没有表现出HEPA 1-6特异性的细胞毒作用。未处理的小鼠和IL-12处理的小鼠的脾细胞也没有显示出细胞毒作用。免疫组织化学分析显示，融合细胞和IL-12处理的小鼠肝内可见T细胞的浸润，而融合细胞处理的小鼠未见。结论：树突状细胞与HEPA 1-6融合细胞免疫小鼠可激发细胞毒性T细胞，识别体内HEPA 1-6与肝细胞共有的表位，但不足以引起肝细胞损伤。IL-12可能参与了该模型肝内CTL浸润和肝细胞损伤的机制。较少

项目成果

Gotaro Toda, Mikio Zeniya et al.: "Autoimmune hepatitis in Japan : epidemiology and clinical features."pp 79-85, Progress in Hapatology, Volume 5 Liver and Immunology Ed. M.Yamanaka, G.Toda, T.Tanaka. Elsevier. (1999)

Gotaro Toda、Mikio Zeniya 等人：“日本自身免疫性肝炎：流行病学和临床特征。”第 79-85 页，《肝脏病学进展》，第 5 卷肝脏和免疫学版。

Mikio Zeniya: "Autoantibodies to hepatocytc plasma membrane autoimmune hepatitis : old and new subjects ; what do we need ?"J Gastroenterol. 35. 252-253 (2000)

Mikio Zeniya：“肝细胞质膜自身免疫性肝炎的自身抗体：新旧科目；我们需要什么？”J Gastroenterol。

Mina Sasaki, Katsumi Yamauchi, Katsutosi Tokushige, Etsuko Isono, Tatsuji Komatsu, Mikio Zeniya, Gotaro Toda, and Naoaki Hayashi: "Clinical significance of Autoantibody to Hepatocyte Mcmbrane Antigen in Type 1 Autoimmune Hepatitis."The Amer J of Gasteroen

Mina Sasaki、Katsumi Yamauchi、Katsutosi Tokushige、Etsuko Isono、Tatsuji Komatsu、Mikio Zeniya、Gotaro Toda 和 Naoaki Hayashi：“1 型自身免疫性肝炎中肝细胞膜抗原自身抗体的临床意义。”Gasteroen 的 Amer J

Mikio Zeniya and Gotaro Toda: "Toward control of hepatitis C in the Asian-Pacific region"J Gastroenterol Hepatol. 15. E117-122 (2000)

Mikio Zeniya 和 Gotaro Toda：“控制亚太地区丙型肝炎”J Gastroenterol Hepatol。

Namiki Izumi, Hiromitsu Kumada, Naoaki Hashimoto, Hideharu Harada, Michio Imawari, Mikio Zeniya and Gotaro Toda: "Rapid Decrease of Plasma HCV-RNA in Early Phase of Twice Daily Administration of 3 MU Doses Interferon-beta in Patients with Genotype lb Hepa

Namiki Izumi、Hiromitsu Kumada、Naoaki Hashimoto、Hideharu Harada、Michio Imawari、Mikio Zeniya 和 Gotaro Toda：“基因型 lb Hepa 患者每日两次服用 3 MU 剂量的干扰素-β，早期血浆 HCV-RNA 快速下降