HBsAg体外冲击的慢性乙肝患者树突状细胞的生物学特性及其对HBV特异性CTL的诱导作用

The biologic character and inducing to the HBV-specific cytotoxic T lymphocytes of DCs from the patients with chronic hepatitis B pulsed with HBsAg in vitro

目的 :研究HBsAg冲击的慢性乙肝患者单核细胞来源的树突状细胞 (DCs)的功能状况及体外对HBV特异性CTL的诱导作用 ,初步探讨诱导特异性抗HBV细胞免疫的途径。方法 :分离慢性乙肝患者外周血单核细胞 ,以GM CSF +IL 4 +TNF α培养诱导DCs,加入HBsAg冲击以诱导HBV特异性DCs。采用FCM测定细胞表面免疫分子CD1a、CD83、CD86、CD80、CD4 0以及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 ,MTT法测定DC刺激同种异体淋巴细胞增殖的能力 ,LDH法检测DC诱导的患者外周血T细胞对HepG2 2 2 15 (转染HBVDNA)、HepG2肝癌细胞株及K5 6 2白血病细胞株的细胞毒作用。结果 :HBsAg冲击的DC其表达CD1a、CD83、CD86、CD80、CD4 0、HLA DR表面分子明显高于对照组 (P <0 0 1,P <0 0 5 ) ,分泌IL 12的水平也高于对照组 (P <0 0 1) ,而分泌IL 6的水平则较对照组显著降低(P <0 0 1) ;HBsAg冲击的DC刺激同种异体淋巴细胞增殖的能力明显增强 (P <0 0 5 ) ,并可有效地诱导自体CTL对转HBV基因的HepG2 2 2 15细胞高效特异性杀伤作用 (P <0 0 1)。结论 :慢性乙型肝炎患者单核细胞来源的DCs经HBsAg抗原冲击后 ,生物学活性增强 ,并且能有效地诱导对HBV特异性反应的CTL。

Objective:To study the functional status of the monocytes-derived dendritic cells (DCs) plused with HBsAg from patients with chronic hepatitis B and their capacity of inducing the HBV- special cytotoxic T lymphocytes (CTLs),and explore a new method of inducing the special anti-HBV cell-mediated immunity.Methods:Monocytes of patients were isolated from peripheral blood and incubated with GM-CSF+IL-4+TNF-α to induce DCs generation. Those DCs were pulsed with HBsAg to induce HBV- special DCs. The phenotype of DCs including CD1a,CD80,CD83,CD86,CD40 and HLA-DR was characterized by FCM and the stimulating reaction of allogenic T lymphocytes was detected by MTT assay. The concentration of cytokines such as IL-12 and IL-6 in supernate was tested by ELISA and the cytotoxicity of CTLs inducing by the DCs against HepG2 2.2.15 cells,HepG2 cells and K562 cells were detected by LDH assay.Results:The expression of CD1a, CD80, CD83,CD86, CD40, HLA-DR molecules on the DCs pulsed with HBsAg was higher than those of the control group (P<0 01, P<0 05). The concentration of IL-12 was higher ( P <0 01),but that of IL-6 was much lower (P<0 01) in the supernatants of the DCs plused with HBsAg. The capacity of DCs pulsed with HBsAg to stimulate proliferation of the allogeneic lymphocyte was increased in comparison with those of controls (P<0 01) and DCs pulsed with HBsAg could induce T lymphocytes to differentiate into HBV- special CTLs which could efficiently kill HepG2 2.2.15 cells that express HBsAg on the surface.Conclusion:By pulsed with HBsAg, the monocytes-derived DCs from patients with chronic hepatitis B can not only increase its biologic activities but efficiently induce HBV special CTLs.