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

1)采用流式细胞术分析各种肝病患者循环树突状细胞亚群。新鲜分离的外周血单核细胞（PBMC）用与异硫氰酸荧光素（FITC）、藻红蛋白（PE）偶联的CD11c或CD123、PerCP偶联的抗HLA-DR和APC偶联的CD40或CD86偶联的谱系（lin）混合物（抗CD3、CD14、CD16、CD19、CD20、CD56）染色。在FACS calibur上分析细胞。DC定义为lin-HLA-DR+细胞，计数CD11c + DC（DC 1）和CD123 + DC（DC 2）的百分比和绝对数。与正常对照组相比，肝病患者循环树突状细胞亚群（DC 1和DC 2）的数量和百分比均减少。此外，随着肝病从慢性肝炎向肝硬化的进展，DC1趋于减少，DC2趋于增加。具有较高活性的肝炎显示外周血中的DC1细胞较少，而肝脏中的DC1细胞较多，这表明DC1被积极招募到肝脏，可能是肝脏中的炎症反应所吸引。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。我们仍在尝试用其他技术如电融合建立杂交瘤。3)将1 × 10^8个细胞输注NOD/SCID小鼠，重建小鼠的免疫系统，结果发现输注2周后，输注小鼠外周血中60%以上的单个核细胞为人PBL，表明CD45阳性。人T、B细胞主要分布于肠系膜淋巴结，血清中可检测到人IgG、IgM和IgA。然而，这些小鼠的疫苗接种尚未成功进行。

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.