外周血PBMC来源的树突状细胞的快速无血清培养方法及细胞信号转导机制  

Rapid serum-free culture of dendritic cells from human peripheral blood monocytes and their intracellular signal transduction

作　　者：吴军[1] 王晓怀[2] 杨德懋[2] 杨太成[2] 王捷[2] 陈政良[1] 

机构地区：[1]南方医科大学免疫学教研室,广东广州510515 [2]广州军区广州总医院肿瘤分子生物学研究所,广东广州510010

出　　处：《第一军医大学学报》2004年第11期1263-1266,共4页Journal of First Military Medical University

基　　金：广东省科技计划项目(20034286)~~

摘　　要：目的探讨体外无血清条件下诱导人外周血单核细胞(PBMC)迅速生成树突状细胞(DC)的方法,并初步探讨钙离子载体(CI)诱导分化的信号转导途径与肿瘤坏死因子(TNF)-α所诱导的是否相同。方法分离健康献血者的PBMC,给予无血清培养基及50 ng/ml的rhGM-CSF过夜培养后,再分别给予100 ng/ml的A23187或50 ng/ml的TNF-α,或预先加入0.5 μg/ml的环胞菌素A(CsA)30 min后,再加入A23187、TNF-α,共培养40 h。于相差显微镜下观察细胞形态的变化,流式细胞仪检测细胞的表面标志,MTT比色法检测不同方法处理的PBMC刺激同种异体T细胞的增殖作用。结果健康献血者的PBMC在无血清条件下,给予rhGM-CSF及CI或TNF-α培养40 h,均可获得DC的典型形态,包括CD14表达下调、CD83及共刺激分子(CD80、CD86)表达上调,以及较强刺激同种异体T细胞增殖的作用;上述由CI诱导的细胞形态的改变、表面分子的表达以及刺激T细胞增殖的作用均可被CsA所抑制。而TNF-α所诱导的细胞形态的改变、表面分子的表达以及刺激T细胞增殖的作用均不受CsA影响。结论健康献血者的PBMCs在体外无血清条件下,可以被rhGM-CSF及CI或TNF-α迅速诱导成DC,但CI与TNF-α诱导PBMC分化为DC的细胞信号转导途径不同。Objective To explore the methods for rapid in vitro culture of the dendritic cells (DCs) from human peripheral blood monocytes (PBMCs) under serum-free conditions and ascertain whether intracellular signal transduction pathway differs between calcium ionophore (CI) and tumor necrosis factor (TNF)-α during their induction of dendritic cell differentiation. Methods PBMCs isolated from healthy donors were plated in serum-free medium supplemented with 50 ng/ml rhGM-CSF. Cells cultured overnight were induced to differentiate with 100 ng/ml A23187 or 50 ng/ml TNF-α, given before or 30 min after pre-treatment with 0.5 μg/ml cyclosporine A (CsA). After culture for 40 h, the cell morphology was observed under phase-contrast microscope, and the surface markers on treated PBMCs were analyzed by flow cytometry. MTT colorimetry was employed to assess the proliferation of the allogeneic T cells. Results PBMCs of healthy donors treated with 50 ng/ml rhGM-CSF in combination with 100 ng/ml CI or 50 ng/ml TNF-α for 40 h exhibited typical morphology of DCs with rapidly decreased CD14 expression and increased expressions of CD83 and co-stimulatory molecules (CD80 and CD86), showing also enhanced ability of stimulating allogeneic T cell proliferation. Calcineurin antagonist CsA inhibited the differentiation induced by CI, but not that induced by TNF-α. Conclusions Under serum-free conditions, both CI and TNF-α are capable of inducing rapid DC differentiation from human PBMCs, but the intracellular signal transduction of CI-induced differentiation is different from that induced by TNF-α.

关 键 词：外周血 树突状细胞 细胞培养 细胞信号 信号转导 单核细胞 肿瘤坏死因子 钙离子载体 

分 类 号：R392.3[医药卫生—免疫学]