HBV表面抗原和核心抗原联合负载的HBV相关性肝癌患者树突状细胞功能研究

摘要

目的 研究HBV相关性肝癌(HCC)患者单核细胞来源的树突状细胞(MoDC)联合负载HBV表面抗原(HBsAg)和HBV核心抗原(HBcAg)后表型和功能的变化.方法 从20例HBV感染相关性HCC患者外周血单个核细胞(PBMC)中诱导出未成熟的MoDC(imaDC),HBcAg与HBsAg联合负载后用"细胞因子鸡尾酒"(IL-1β、ID-6、TNF-α和PGE2)促成熟,流式细胞仪检测成熟MoDC的表型,酶联免疫吸附试验(ELISA)检测MoDC分泌的细胞因子,酶联免疫斑点(Elispot)实验检测MoDC诱导自身T细胞分泌IFN-γ的频率,五聚体染色法检测MoDC诱导自身T细胞产生HBV特异性CD8 T细胞的能力.结果 HBsAg和HBcAg联合负载的成熟MoDC(scDC)表面分子表达水平比imaDC显著增加,其中HBsAg、抗-HBe、抗-HBc三项阳性(简称为1-4-5阳性组)的患者MoDC表面分子明显高于HBsAg和抗-HBc两项阳性(简称为1-5阳性组)的患者;scDC分泌IL-12和IL-10水平明显高于用无关蛋白负载的对照组(conDC),1-4-5阳性组患者MoDC分泌的IL-12水平明显高于1-5阳性组;1-4-5阳性组的scDC诱导自体T细胞分泌IFN-γ的频率明显高于conDC;有4例HLA-A2+的1-4-5阳性组患者的scDC诱导自体T细胞产生HBVcorel8-27特异性CD8 T细胞.结论 HBsAg和HBcAg联合负载可以逆转HBV相关性HCC患者MoDC受损的功能,增强1-4-5阳性组MoDC;诱导自体T细胞特异性应答的能力.1-4-5阳性组患者的MoDC功能强于1-5阳性组患者.%Objective To investigate the characteristics of HBsAg and HBcAg in combination pulsed monocyte-derived DCs(MoDC) derived from patients with HBV-associated hepatocellular carcinoma(HCC).Methods MoDCs were generated from 20 HBV-associated HCC patients,and pulsed with recombinant human serum albumin(rHSA)as conDC(control DC),or pulsed with HBsAg andHBcAg in combination as scDC.Phenotypic patterns of MoDCs were characterized by flow cytometry,and the levels of cytokines releasedby MoDCs were analyzed by ELISA,and frequencies of IFN-γ-producing antigen-specific autologous T cells were measured by Elispotassay.HBV-specific CD8 T cells proliferation was determined by pentamer staining.Results 1.The levels of MHC and costimulatorymolecules expressed on scDC were significantly higher than those on immature MoDC(imaDC)in two groups,and the levels of MHC andcostimulatory molecules expressed on imaDC,conDC and scDC in HBsAg+,anti-HBe+,anti-HBc+patients(1-4-5 positive group)were significantly higher than those in HBsAg+,anti-HBc+patients(1-5 positive group);2.The levels of IL-12 and IL-10 produced byscDC were higher than those produced by imaDC and conDC in both groups,and the levels of IL-12 produced by imaDC,conDC and scDCin 1-4-5 positive group were higher than those in 1-5 positive group.3.The frequencies of IFN-γ-producing T cells induced by scDC werehigher than those by conDC in 1-4-5 positive group.4.scDCs from 4 cases of HLA-A2+patients in 1-4-5 positive group could induceautologous T cells to generate HBVcore18-27-specific CD8 T cells.Condusions HBsAg and HBcAg pulse in combination couldsubstantially reverse the impaired function of MoDCs in HBv-associated HCC patients,and boost MoDC to induce HBV-specific T cellsresponse,especially in HBsAg+,anti-HBe+ and anti-HBe+ patients.