INDUCTION OF HUMAN HEPATOMA-SPECIFIC IMMUNE RESPONSE BY HEPATOMA-DENDRITIC CELL HYBRIDOMAS

肝癌-树突状细胞杂交瘤诱导人肝癌特异性免疫反应

• 批准号: 11670485
• 负责人: SHIMIZU Yukihiro
• 金额: $ 0.9万
• 依托单位: TOYAMA MEDICAL AND PHARMACEUTICAL UNIVERSITY
• 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 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670485/
• 关键词: liver disease; dendritic cells; hybridomas; SCID mice; NOD; huマウス; フローサイトメトリー; CD11c; CD123; 共刺激分子; SCID-hu マウス

1) Circulating dendritic cell subsets were analyzed by flow cytometry in patients with various liver diseases. Freshly-isolated peropheral blood mononuclear cells (PBMNCs) were stained with a lineage (lin) cocktail (anti-CD3, CD14, CD16, CD19, CD20, CD56) conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE)-conjugated CD11c or CD123, PerCP-conjugated anti-HLA-DR, and APC-conjugated CD40 or CD86. The cells were analyzed on a FACS calibur. DCs were defined as lin-HLA-DR+ cells, and the percentages and absolute numbers of both CD11c+DC (DC1) and CD123+DC (DC2) were counted. The numbers and percentages of circulating dendritic cell subsets (both DC1 and DC2) were fewer in patients with liver diseases compared with nornal controls. Moreover, as liver disease progress from chronic hepatitis to liver cirrhosis, DC1 tended to decrease and DC2 increased. Hepatitis with higher activity showed fewer DC1 cells in peripferal blood and more those cells in the liver, suggesting that DC1 are actively recruited to the liver, possibly chemoattracted by the inflammatory response in the liver. 2)All the attempts to establish the human hepatoma-dendritic cell hybridomas have been failed. We are stll trying to establish the hybridomas using other techniques such as an electroric fusion. 3) Reconstruction of human immune system in the mice was attempted by infusion of 1×10 ^8 cells into NOD/SCID mice, and we found that more than 60% of mononuclear cells in peripheral blood of the infused mice were human PBL 2 weeks after infusion, indicated by CD45 positivity. Human T and B cells were mainly found in lymph nodes in mesenteruim, and human IgG, IgM and IgA were detected in the sera of those mice. However, vaccination in those mice has not been successfully performed, yet.

1)用流式细胞术分析各种肝脏疾病患者的循环树突状细胞亚群。用异硫氰酸荧光素（FITC）、植红蛋白（PE）偶联的CD11c或CD123、过磷酸基偶联的抗hla - dr和apc偶联的CD40或CD86对新分离的外周血单核细胞（pbmnc）进行染色（抗cd3、CD14、CD16、CD19、CD20、CD56）。用流式细胞仪对细胞进行分析。将DC定义为lin-HLA-DR+细胞，计数CD11c+DC （DC1）和CD123+DC （DC2）的百分比和绝对数量。与正常对照相比，肝脏疾病患者循环树突状细胞亚群（DC1和DC2）的数量和百分比更少。此外，随着肝病从慢性肝炎发展到肝硬化，DC1呈下降趋势，DC2呈上升趋势。活动性较高的肝炎患者外周血中的DC1细胞较少，而肝脏中的DC1细胞较多，这表明DC1被积极募集到肝脏，可能是肝脏炎症反应的化学吸引。2)所有建立人肝癌-树突状细胞杂交瘤的尝试均以失败告终。我们仍在尝试使用其他技术，如电子聚变，来建立杂交瘤。3)通过向NOD/SCID小鼠输注1×10 ^8细胞，尝试重建小鼠的人免疫系统，我们发现输注后2周小鼠外周血中60%以上的单核细胞为人PBL， CD45阳性。人T、B细胞主要分布于肠系膜淋巴结，血清中检测到人IgG、IgM、IgA。然而，在这些小鼠中接种疫苗尚未成功。