摘要
目的：研究HBsAg抗原冲击对慢性乙型肝炎病人外周血单核细胞来源的、体外培养诱导的树突状细胞(Dendritic Cell, DC)的免疫分子表达、免疫功能以及激发抗HBsAg特异性细胞免疫的影响，初步探讨以DC为免疫佐剂，体外激发高特异性抗HBV细胞免疫的途径，从而为慢性乙型肝炎免疫治疗提供新依据。方法：分离慢性乙型肝炎患者外周血单核细胞，以GM-CSF(800u／ml)+IL-4(500u／ml)+TNF-α(250u／ml)细胞因子组合诱导的DC，于培养第4天将培养的DC分成两组，一组加入纯化的HBsAg(10ng／ml)抗原进行冲击，称为实验组，另外一组的DC按常规方法继续培养称之为对照组。两组均于第7天，以FACS测定细胞表面免疫分子CDla、CD83、CD86、CD80、CD40、HLA—DR的表达水平，ELISA法检测培养上清中细胞因子IL-6、IL-12的分泌含量及DC刺激同种异体淋巴细胞增殖能力。本实验还将诱导的DC于培养的第4天以纯化的HBsAg进行冲击，第5天与自体T细胞共培养，第7天再次用抗原冲击，在第10天采用LDH法分别检测T细胞对HepG2 2.2.15、HepG2肝癌细胞株及K562白血病细胞株的细胞毒作用。结果：(1)HBsAg冲击的实验组DC其CDla、CD83、CD86、CD80、CD40、HLA—DR的表达与未冲击HBsAg的DC比较有明显增高(p＜0.01)；(2)实验组DC培养上清IL—12的浓度高于对照组(P＜0.01)，而IL—6的浓度则较对照组显著降低(P＜0.01)；(3)实验组DC刺激同种异体淋巴细胞增殖的能力较对照组明显增强(P＜0.05)；(4)HBsAg冲击的DC可有效地诱导自体CTL对转HBV基因的HepG2 2.2.15细胞有高效杀伤作用，HBsAg+DC+T组杀伤率为66.0％±2.7％，较DC+T及HBsAg+T组明显增高(P＜0.01)，各组对肝源性HepG2及非肝源性K562细胞的非特异性杀伤作用则较弱。结论：慢性乙型肝炎患者外周血单核细胞经GM—
HBsAg冲击对慢性肝炎患者外周血来源DC的作用及激发cTL的效应摘要
esF+IL一4+TNF一a诱导的DC经HBsAg抗原冲击后，CDla、CD83、
CD86、CD8o、CD4o、HLA一DR等分子呈上调性表达，，刺激同种异
体淋巴细胞增殖的能力增强，分泌的IL一12明显增高，分泌IL一6明
显降低，而且其诱导的HBV特异性CTL杀伤作用显著增强。本实验
结果提示:慢性乙型肝炎患者外周血来源的单核细胞经细胞因子诱导
的Dc经HBsAg冲击后，能激发高效HBsAg特异的CTL杀伤作用，
为肝炎病人抗病毒的细胞免疫治疗提供实验依据。
Objective: To study the function including immunophenotypic, immunologic function and anti-HBsAg specific cell-mediated immunity of monocytes-derived dendritic cell(DCs) plused with HBsAg from patients with chronic hepatitis B. To explore a new method that the DCs plused with HBsAg are used as immunologic adjuvant to get an efficient and HBsAg-special CTL response. Methods: DCs generating from periopheral blood monocytes are cultured in media containing rhGM-CSF(800u/ml), rhIL-4(500u/ml)and rhTNF- a (250u/ml). Then the DCs divided into two groups, one was the experimental group plused with HBsAg(10ng/ml)on the 4th day ; the other was the control group without HBsAg. On the 7th day, immunophenotype of DCs in the two groups including CD1a, CD80, CD83, CD86, CD40, HLA-DR was characterized by FACS and immunologic function of them was detected through the stimulating reaction of allogenic T lymphocytes. The concentration of cytokines in supernatant such as IL-6 and IL-12 was tested by ELISA .In the experiment, HB
sAg were added into the DCs on the 4th day,then the DCs were co-cultured with T cells on the 5th day .On the 7th day, HBsAg was added into media. On the 10th day ,the CTL's response to HepG2 2.2.15cells,HepG2 cells and K562 cells were detected by LDH. Results: (l)Expression of the immunophenotype of the experimental groups including CD1a, CD80, CD83, CD86, CD40, HLA-DR was higer than that of the control group (p<0.01) (2)The concentration of IL-12 in the supernatant of the experimental groups was
higer and IL-6 was much lower than that of the control groups (p<0.01)(3)The capacity of DCs stimulating allogenic lymphocyte to proliferate greatly was increased compared with that of control(p<0.01) (4)DCs plused with HBsAg could efficiently present HBsAg and induce T lymphocytes to differentiate into CTLs, which could efficiently kill HepG2 2.2.15cells that express HBsAg on the surface .The killing rate of the experimental groups (66.0% +2.7%) was much higer than that of the two control groups (p<0.01).But CTLs of the three groups have quite lower ability to kill hepatogenic HepG2 cells and non- hepatogenic K562 cells. Conclusion: DCs plused with HBsAg, which developed with rhGM-CSF, rhIL-4 and rhTNF- a , could greatly increase expression of immunophenotype of DCs (CD1a, CD80, CD83, CD86, CD40, HLA-DR). Meanwhile, DCs still enchance the capacity of stimulating allogenic lymphocyte to proliferate. The level of IL-12 secretion increased,but the level of IL-6 secretion decreased. Specical and efficient CTL r
esponse can be significantly induced by DCs plused with HBsAg. It will probably provide a experiemental evidence as immunotherapy for patients with Chronic hepatitis B.
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