摘要
目的研究慢性乙肝合并肝细胞癌(HCC)患者外周血髓样树突状细胞(mDC)负载乙型肝炎病毒(HBV)抗原后表型和功能的变化。方法用成分血分离机采集21例肝细胞癌患者和4名健康人的外周血单核细胞(PBMC),用无血清AIMV培养基加入粒细胞巨噬细胞集落刺激因子(GMCSF)和白细胞介素(IL)4等因子诱导mDC分化、发育和成熟,同时加入HBcAg或HBsAg致敏mDC,通过流式细胞仪分析mDC表面分子的表达,用酶联免疫吸附试验(ELISA)检测mDC培养上清中IL12、IL10的含量,通过自体混合淋巴细胞反应(AMLR)比较抗原致敏后mDC刺激淋巴细胞增殖的能力。结果在肝细胞癌患者中,(1)经HBcAg致敏的mDC表面分子CD80、CD86、CD40、HLADR的表达率(55%±26%、80%±13%、70%±13%、73%±24%)明显高于无抗原致敏组mDC(29%±25%、35%±18%、44%±26%、45%±23%,P<005);经HBsAg致敏的mDC仅HLADR的表达率(63%±15%)高于无抗原致敏组(45%±23%,P<005),其他表面分子差异无统计学意义。(2)在AMLR中经HBV抗原致敏的mDC刺激淋巴细胞增殖的能力明显高于无抗原致敏组(P<005),而且HBcAg的致敏效果优于HBsAg的致敏效果(P<005)。(3)经HBcAg致敏的mDC分泌IL10和IL12的量(236pg/ml±95pg/ml,733pg/ml±212pg/ml)明显高于经HBsAg致敏的mDC(35pg/ml±9pg/ml。
Objective To investigate the characteristics of phenotype and function of myeloid dendritic cells (mDCs) pulsed with HBV antigens derived from HBV-associated hepatocellular carcinoma (HCC) patients. Methods Peripheral blood mononuclear cells (PBMCs) were collected, using blood cell separator, from 21 primary HCC patients and 4 healthy donors. mDCs were propagated in serum-free AIM-V medium in the presence of cytokine cocktail, and pulsed with HBcAg or HBsAg. After 9 days' incubation, the phenotypic patterns of mDC were characterized by flow cytometry and the levels of IL-10 and IL-12 produced by mDCs were analyzed by ELISA. Autologous T cells proliferation stimulated by mDC was tested by non-radioactive cell proliferation assay kit. Results The expression rates of CD80, CD86, CD40, and HLA-DR in the mDCs pulsed with HBcAg were 55%±26%,80%±13%,70%±13% and 73%±24% respectively, significantly higher than those of the control group (29%±25%,35%±18%,44%±26% and 45%±23% respectively, all P<0.05). Among the expression rates of surface molecules of the mDCs pulsed with HBsAg only the expression rate of HLA-DR was significantly higher than that of the un-pulsed mDCs (63%±15% vs 45%±23%, P<0.05). T cell proliferation assay revealed an impaired allostimulatory capacity of mDCs in HCC and the stimulatory capacity of the mDCs pulsed with HBcAg to induce proliferation of autologous T cells was more powerful than that pulsed with HBsAg (P<0.05). The levels of IL-10 and IL-12 produced by the mDCs pulsed with HBsAg were (35 pg/ml±9 pg/ml and 135 pg/ml±63 pg/ml respectively, both significantly lower than those pulsed with HBcAg (236 pg/ml±95 pg/ml and 733 pg/ml±212 pg/ml respectively, both P<0.05). Conclusion Pulsation of mDCs in vitro by HBcAg or HBsAg enhance the expression of CD80, CD86, CD40, and HLA-DR, and increase the ability of mDCs to stimulate the proliferation of autologous T lymphocytes.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2005年第4期248-252,共5页
National Medical Journal of China
基金
国家自然科学基金资助项目(30271230
30371300)
