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

南方医科大学学报 ›› 2004, Vol. 24 ›› Issue (11): 1263-1266.

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

吴军¹, 王晓怀², 杨德懋², 杨太成², 王捷², 陈政良¹

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

出版日期:2004-11-20 发布日期:2004-11-20
基金资助:
收稿日期:2003-11-8。
基金项目:广东省科技计划项目(20034286)
作者简介:吴军(1967- ),女,南方医科大学在读博士研究生,主治医师,电话:020-36222205-53479,E-mail:Juwuwang@Hotmail

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

WU Jun¹, WANG Xiao-huai², YANG De-mao², YANG Tai-cheng², WANG Jie², CHEN Zheng-liang¹

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

Online:2004-11-20 Published:2004-11-20

摘要/Abstract

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。

Abstract: 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.Conclusion s 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.

中图分类号:

R392.3

吴军¹, 王晓怀², 杨德懋², 杨太成², 王捷², 陈政良¹. 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[J]. 南方医科大学学报, 2004, 24(11): 1263-1266.

WU Jun¹, WANG Xiao-huai², YANG De-mao², YANG Tai-cheng², WANG Jie², CHEN Zheng-liang¹. Rapid serum-free culture of dendritic cells from human peripheral blood monocytes and their intracellular signal transduction[J]. Journal of Southern Medical University, 2004, 24(11): 1263-1266.

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