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目的初步探讨钙离子载体(CI)在体外迅速诱导人外周血单核细胞(PBMC)分化为树突状细胞(DC)的信号转导途径。方法分离健康献血者的PBMC,在体外用人重组粒/巨噬细胞集落刺激因子(rhGMCSF)和CI培养40h或rhGMCSF和TNFα培养5d,部分PBMC用环胞菌素A(CsA)预处理30min后,再加入CI或TNFα;相差显微镜下观察细胞的形态;流式细胞仪检测细胞表面CD14、CD80、CD86、CD83、HLADR等分子的表达;MTT比色法检测其对同种异体T淋巴细胞的刺激增殖作用;凝胶电泳迁移率变动分析(EMSA)检测不同方法培养的细胞其核转录因子κB(NFκB)的活化水平。结果健康献血者的PBMC经rhGMCSF与CI培养40h或rhGMCSF与TNFα培养5d,均可获得DC的典型形态和表面分子的表达,包括CD14表达下调,CD80、CD86及HLADR等分子表达的上调,以及较强刺激同种异体T淋巴细胞增殖的作用;其中CI诱导的DC其CD80、CD86、CD83、HLADR等分子的上调更明显,刺激T淋巴细胞的增殖能力更强。经TNFα及CI所诱导分化的DC均具有较好的NFκB活性。但经CI诱导的DC,其形态、表面标志物、对T淋巴细胞的刺激增殖能力及NFκB的活性,均受到CsA的抑制;而TNFα所诱导的DC却不受CsA的影响。结论CI比TNFα更迅速、更高效地诱导PBMC向DC分化的原因,是信号转导途径的不同,但不论上游信号转导途径有何不同,两者最终都通过激活NFκB来诱导细胞的分化。
Objective To investigate the signal transduction pathways of calcium ion (CI) cells rapidly inducing the differentiation of human peripheral blood mononuclear cells (PBMCs) into dendritic cells (DCs) in vitro. Methods PBMC from healthy blood donors were isolated and cultured in vitro. Human peripheral blood mononuclear cells (rhGMCSF) and rhGMCSF were incubated with rhGMCSF for 40 h or rhGMCSF for 5 days. PBMCs were pretreated with CsA for 30 min. Then add CI or TNFα; observe the morphology of cells by phase contrast microscopy; detect the expression of CD14, CD80, CD86, CD83, HLADR and other molecules on the cell surface by flow cytometry; and stimulate the allogeneic T lymphocytes by MTT assay Proliferation and the activity of nuclear factor kappa B (NFκB) in cells cultured by different methods were detected by electrophoretic mobility shift assay (EMSA). Results The typical morphology and surface molecule expression of DC in healthy donors PBMCs cultured with rhGMCSF and CI for 40h or rhGMCSF and TNFα for 5 days were down-regulated, including upregulation of CD14, up-regulation of CD80, CD86 and HLADR, Strongly stimulated the proliferation of allogeneic T lymphocytes; among them, CI induced DCs had more obvious up-regulation of CD80, CD86, CD83, HLADR and other molecules, and stimulated the proliferation ability of T lymphocytes stronger. DCs induced by TNFα and CI had better NFκB activity. However, CsA-induced DCs were inhibited by CsA for their morphology, surface markers, T-lymphocyte proliferation and NFκB activity, whereas DCs induced by TNFα were not affected by CsA. CONCLUSION: The reason that CI induces differentiation of PBMC into DC more rapidly and efficiently than TNFα is due to the different signaling pathways, however, both ultimately induce cell differentiation by activating NFκB, regardless of the difference in upstream signaling pathways.