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目的:体外诱导、培养单核细胞源性树突状细胞(DC),研究其淋巴细胞趋化因子(lymphotactin,Lptn)mRNA表达的动态变化。方法:采用密度梯度离心的方法分离人外周血中的单个核细胞(PBMC),用重组人粒细胞-巨噬细胞集落群体刺激因子(rhGM-CSF)、重组人白细胞介素-4(rhIL-4)刺激贴壁的单核细胞,诱导培养DC,第6天用重组人肿瘤坏死因子-α(rhTNF-α)诱导DC成熟。用流式细胞术检测成熟和未成熟的DC表面分子CD1a和CD83;在电镜下观察成熟DC的形态;以RT-PCR法扩增其LptncDNA并克隆至pGM-TEasyT载体中,测序;以RT-PCR结合凝胶成像分析系统,半定量分析培养3、5及7dDC的LptnmRNA表达的强度。结果:电镜观察培养7d的细胞具有典型的DC形态,流式细胞术检测DC表面分子CD83呈高水平表达。用RT-PCR法克隆的cDNA序列与GenBank中U23772(登陆号)提供的序列一致。培养3d的DC不表达LptnmRNA,培养7d的DC较培养5d的DCLptnmRNA表达增强。结论:单核细胞源性DC能表达LptnmRNA,随着DC的成熟,LptnmRNA的表达增强。
OBJECTIVE: To induce and culture monocyte-derived dendritic cells (DCs) in vitro and study their dynamic changes of lymphotactin (Lptn) mRNA expression. Methods: Mononuclear cells (PBMCs) were isolated from human peripheral blood by density gradient centrifugation. Recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF), recombinant human interleukin-4 (rhIL- 4) Adherent monocytes were stimulated to induce DCs and DC maturation induced by recombinant human tumor necrosis factor-α (rhTNF-α) on the 6th day. The matured and immature DC surface molecules CD1a and CD83 were detected by flow cytometry. The morphology of mature DCs was observed under electron microscope. The Lptn cDNA was amplified by RT-PCR and cloned into pGM-TEasyT vector and sequenced. PCR combined with gel imaging analysis system to semi-quantitatively analyze the intensity of LptnmRNA expression in cultured 3,5 and 7dDC. Results: The cells cultured for 7 days under electron microscope showed typical DC morphology. The expression of CD83 on DCs was high level by flow cytometry. The cDNA sequence cloned by RT-PCR was identical to the sequence provided in GenBank U23772 (accession number). DCs cultured for 3 days did not express LptnmRNA, DCs cultured for 7 days increased DCLptnmRNA expression after cultured for 5 days. CONCLUSION: Monocyte-derived DC can express LptnmRNA, and with the maturation of DC, LptnmRNA expression is enhanced.