慢性乙型肝炎病人外周血树突状细胞功能的研究

目的：体外培养慢性乙型肝炎病人的树突状细胞，并从细胞表型和功能方面与正常人树突状细胞进行比较。方法：从慢性乙型肝炎病人及正常人外周血分离单个核细胞，加入含1000U／ml GM-CSF和500U/ml IL-4的无血清培养基AIM－V体外培养，获得树突状细胞。利用流式细胞仪检测细胞因子TNF－α对树突状细胞培养的影响，IL－12ELISA试剂盒检测树突状细胞分泌IL－12的水平，并采用MLR方法检测树突状细胞刺激同种异体T淋巴细胞增殖的能力。结果：1．慢性乙型肝炎病人的外周血单个核细胞用AIM－V培养及细胞因子诱导后，可获得成熟的具有典型形态的树突状细胞，加入TNF－α后，IL－12分泌增高，CD83表达增加；2．病人树突状细胞与正常人比较，CD80表达较正常人的低（P＜0．05），刺激同种异体T细胞增殖能力低于正常人。结论：1．慢性乙型肝炎病人的树突状细胞与正常人一样可用AIM－V无血清培养基及特定的细胞因子诱导在体外大量获得；2．慢性乙型肝炎病人的树突状细胞与正常人相比较在功能上降低，在形态数量上差异无显著意义。

Functions of cultured dendritic cells from patients with chronic hepatitis Beta decreased

OBJECTIVE: To compare the phenotype and function of dendritic cells (DCs) of patients with chronic hepatitis Beta with those of DCs from healthy persons. METHOD: Peripheral blood monocytes (PBMCs) were isolated from 10 patients with chronic hepatitis Beta and 10 healthy blood donors. DCs were generated by culturing PBMCs in 1 000 U/ml granulocyte-macrophage stimulating factor (GM-CSF) and 500 U/ml IL-4 with AIM-V in vitro. DCs and supernatant were collected 3, 5, 7, 9, 11, 13 and 15 days after culture. TNF- alpha 50 ng/ml was added into the culture of DCs from patients with hepatitis Beta and DCs were collected 48 hours after. FACS was used to detect the phenotype. The changes of cell phenotype on different days with or without TNF-alpha were tested by immunolabelling and flow cytometry analysis. IL-12 ELISA kit was applied to investigate IL-12 secretion of DCs. To perform mixed leucocyte reaction (MLR) test, DCs were cocultured with allogeneic T cells at different stimulatory ratio. RESULTS: The expression of CD83 was higher in the group with TNF-alpha than in the group without TNF-alpha. The positive rates of CD80 and CD54 were not different between the two groups. DCs from patients and normal donors with typical morphology were harvested successfully in vitro. The IL-12 level was 103.93 +/- 12.59 pg/ml in the group with TNF-alpha, higher than that in the group without TNF-alpha (11.45 +/- 2.58 pg/ml, P < 0.05). MLR test showed that when the stimulatory ratio of allogeneic T cells was 1 : 20, 1 : 50, and 1 : 100 respectively, the results were 40 835 +/- 3 480, 37 525 +/- 6 398, and 31 814 +/- 5 521 respectively in group of patient; and 49 097 +/- 2 273, 43 049 +/- 2 833, and 39 091 +/- 6 469 respectively in the group of healthy blood donors. CONCLUSION: 1.DCs of patients and of healthy persons can be cultured success fully with AIM-V when induced by GM-CSF, IL-4 and TNF-alpha in vitro. 2. Functions of DCs from patients are lower than those from healthy donors. However, they are not different in amount and morphology.