乙型肝炎相关肝癌外周血树突状细胞负载肿瘤抗原前后免疫功能的变化

Changes in the immune function of dendritic cells (DC) derived from HBV-related hepatocellular carcinoma (HCC) patients' peripheral blood monocytes (PBMC) pulsed with tumor antigen

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 identify the phenotype and immune function of dendritic cells derived from HBV-related HCC patients' peripheral blood monocytes pulsed with soluble tumor antigen, and their relation to immune escape. Methods Peripheral blood monocytes were isolated from 18 HBV-related hepatocellular carcinoma (HCC) patients,11 HBV-related liver cirrhosis patients (LC) and 10 health blood donors; DCs were induced in the completed medium containing GM-CSF and IL-4. The morphology of DCs was studied using a confocal microscope and scanning electronic microscope, and the phenotype of DCs were detected by flow cytometric analysis. The mixed leucocyte reaction test was employed to determine the stimulatory capacity of DCs before and after being pulsed with soluble tumor antigen (prepared from HCCLM6 cell line). IL-12 ELISA kit was used to investigate IL-12 secretion of DCs in the supernate of MLR. Results The amount of PBMC and DCs was significantly lower in LC and HCC compare to those in the healthy subjects; the expression levels of HLA-DR, CD1a, CD80 and CD86 on DC surfaces were lower in LC and HCC patients than those of the healthy group; the stimulating capacity of DC in MLR and levels of IL-12 in supernate of MLR were also lower in LC and HCC, but were enhanced after tumor antigen pulsed in all three groups, particularly in the LC group; the secretion of IL-12 in MLR supernate was still lower than that of the healthy group. Conclusion The phenotype and function defects of DC derived from PBMC of LC and HCC patients might play a key role in immune escape in HBV infection and HCC. The function of DC of LC patients can be enhanced after the tumor was antigen-pulsed.