树突状细胞体外刺激对HBV特异性细胞毒T细胞影响的研究  

The effect of stimulation of dendritic cell on HBV-epitopic cytotoxic T-lymphocyte

作　　者：张恒辉[1] 何豫[1] 赵鸿[1] 朴文花[1] 刘茂昌[1] 席宏丽[1] 于敏[1] 王贵强[1] 

机构地区：[1]北京大学第一医院感染疾病科,100034

出　　处：《中华医学杂志》2005年第17期1171-1176,共6页National Medical Journal of China

基　　金：卫生部临床学科重点项目基金(20010911);北京大学985青年基金;美国CMB基金项目

摘　　要：目的探讨通过聚肌胞体外作用树突状细胞后改善慢性乙型肝炎患者树突状细胞功能,并在体外活化自身T细胞获得高频数HBV特异性细胞毒性T细胞(CTL)的方法。方法分离慢性乙型肝炎病人外周血单个核细胞,在粒巨噬细胞集落刺激因子(GM CSF)和白细胞介素4(IL4)的诱导下培养树突状细胞。培养的第7天加入聚肌胞刺激获得成熟树突状细胞,经HBVcore1827肽负载后与自身T淋巴细胞共培养,通过酶联斑点计数法(Elispot)及MHC肽四聚体法(Tetramer)比较病人T细胞未经自身树突状细胞刺激组、经HBVcore1827肽负载的自身树突状细胞刺激组、经聚肌胞促成熟的HBVcore1827肽负载的自身树突状细胞刺激组中HBV特异性的CTL的功能和频数。结果慢性乙型肝炎病人外周血单核细胞体外经GM CSF和IL4诱导可转化为树突状细胞,树突状细胞转化过程中聚肌胞的刺激可显著上调树突状细胞表面分子CD80、CD83的表达(P<0.01),促进树突状细胞的成熟。Elispot法检测分泌IFNγ的CTL的频数病人T细胞未经自身树突状细胞刺激组分泌频数为(9～28)/1×105T细胞,均值16;经HBVcore1827肽负载的自身树突状细胞刺激组频数为(30～67)/1×105T细胞,均值为46;经聚肌胞促成熟的HBVcore1827肽负载的自身树突状细胞刺激组频数为(59～130)/1×105T细胞,均值为98。三组数据?Objective To observe the enhancement of cytotoxic T lymphocyte responses in patients with chronic hepatitis B following vaccination with dendritic cell stimulated by Poly (I:C) in vitro. Methods^Peripheral blood mononuclear cells (PBMC) were isolated from whole blood by density gradient centrifugation on Ficoll-Hypaque, the adherent cells were cultured in the medium AIM-V contained recombinant human IL-4 and recombinant human GM-CSF. On day 7, one part of wells were added with Poly (I:C). On day 9, mature DCs (mDCs) were harvested and used to phenotype analysis. Both of the immature DCs and mature DCs were concultured with the autologous T cells for another two-three days. According to the source of dendritic cells concultured with the T cells, the subjects were divided into three groups: the T cells isolated from CHB group; the T cells stimulated by dendritic cells pulsed with HBcAg18-27 CTL epitope peptide group; the T cells stimulated by dendritic cells concultured with Poly (I:C) and pulsed with HBcAg18-27 CTL epitope peptide group. The function and frequency of HBV specific CTL were detected by Elispot (Enzyme-linked immunospot assay, Elispot) and tetramer staining. Results The average of percentage of HBV specific CD8+ cells of total CD8+ cells was 0.77 % (0.45 % ～1.74 %) in the T cells isolated from 22 chronic hepatitis B patients and the average of the spots of antigen-specific IFN-γ-releasing effector cells was 16(9 ～28); The percentage of HBV specific CD8+ cells of the T cells stimulated by dendritic cells pulsed with HBcAg18-27 CTL epitope peptide group rose to 1.92%(1.36%～2.65%) and the average of the spots of antigen-specific IFN-γ-releasing effector cells was 46(30 ～67); while stimulated by dendritic cells added with Poly (I:C) and pulsed with HBcAg18-27 CTL epitope peptide, the percentage of HBV specific CD8+ cells of total CD8+ cells rose to 3.49 % (2.02% ～4.60 %) and the average of the spots was 98(59 ～130). There was statistical difference between the results of Elispot and tetrame

关 键 词：特异性细胞毒T细胞 影响的研究 白细胞介素-4(IL-4) 自身树突状细胞 体外刺激 慢性乙型肝炎患者 巨噬细胞集落刺激因子 慢性乙型肝炎病 HBV特异性 人外周血单个核细胞 Elispot法 TETRAMER 外周血T淋巴细胞 细胞毒性T细胞 

分 类 号：R512.62[医药卫生—内科学]