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目的探讨肝癌患者外周血单个核细胞(PBMC)来源树突状细胞(DC)表面分子表达及负载肿瘤抗原前后免疫功能变化与免疫逃逸的关系。方法分离18例乙型肝炎相关原发性肝癌、11例乙型肝炎肝硬化患者和10名健康献血者PBMC,体外培养,并加入重组人粒细胞巨噬细胞集落刺激因子(GM-CSF)和白细胞介素-4(IL-4)诱导DC。以共聚焦显微镜和扫描电镜观察形态,以流式细胞仪检测DC表面人类白细胞抗原(HLA)-DR、CD1a、CD80、CD83、CD86等分子表达水平。以HCCLM6肝癌细胞株制备肿瘤抗原,分别负载3种DC,最后以混合淋巴细胞反应(MLR)测定DC负载前后刺激同种异型T淋巴细胞增殖能力,并测定MLR上清液中IL-12的含量。结果肝硬化和肝癌组PBMC、DC得率低于正常组(P<0.05);HLA- DR、CD1a、CD80和CD86等分子表达水平也低于正常组(P<0.05);负载肿瘤抗原前肝硬化和肝癌组刺激同种异型T淋巴细胞增殖能力和MLR上清液中IL-12含量明显低于正常组,负载肿瘤抗原后3组均提高, 并以肝硬化组提高最为明显,但IL-12含量仍低于正常组。结论DC表型和功能缺陷可能是乙型肝炎病毒产生免疫耐受和肝癌细胞免疫逃逸的重要机制。肝硬化患者DC仍有一定功能。
Objective To investigate the molecular expression of peripheral blood mononuclear cells (PBMCs) on dendritic cells (DCs) and the relationship between immune function and immune escape before and after tumor antigen loading. Methods PBMC from 18 patients with primary hepatocellular carcinoma associated with hepatitis B, 11 patients with hepatitis B cirrhosis and 10 healthy blood donors were isolated and cultured in vitro. Recombinant human granulocyte macrophage colony stimulating factor (GM-CSF) and Interleukin-4 (IL-4) induces DCs. The morphological changes were observed by confocal microscopy and scanning electron microscopy. The expression of HLA-DR, CD1a, CD80, CD83, CD86 and other molecules on DCs were detected by flow cytometry. The tumor antigens were prepared from HCCLM6 hepatocarcinoma cell lines and loaded with three kinds of DC respectively. The proliferation of allogeneic T lymphocytes was measured before and after DC loading by mixed lymphocyte reaction (MLR), and the content of IL-12 in MLR supernatant . Results The rates of PBMC and DC in cirrhosis and hepatocellular carcinoma were lower than those in normal group (P <0.05). The expression of HLA-DR, CD1a, CD80 and CD86 were also lower than those in normal group (P <0.05) The sclerotic and hepatocellular carcinoma groups stimulated the proliferation of allogeneic T lymphocytes and the levels of IL-12 in MLR supernatant were significantly lower than those in normal group. After loading tumor antigen, the three groups were increased and the most obvious was in cirrhosis group. However, IL- 12 content is still lower than the normal group. Conclusion DC phenotype and functional defects may be an important mechanism of hepatitis B virus immune tolerance and immune escape of liver cancer cells. DC patients with cirrhosis still have some function.