肿瘤坏死因子—α预刺激培养成熟树突状细胞

目的 利用肿瘤坏死因子-α(TNF-α)诱导树突状细胞(DC)成熟的作用原理培养成熟DC的新方法。方法Ficoll密度梯度离心分离健康人外周血获得单个核细胞，用含有10％胎牛血清的RPMI640培养基培养。TNF-α预刺激4h后加入粒细胞巨噬细胞集落刺激因子(GM—CSF)及IL-4，48、96h再次同时加入上述3种细胞因子并继续培养，第8—10天收集细胞，记作T—DC。FCM检测FDC表型，用^3H-TdR检测同、异基因混合淋巴细胞反应(MLR)及抗原提呈功能；MTT法检测FDC诱导T细胞的杀伤活性。结果 T—DC表达CD83、CDla、CD54、CDlla、CD80、CD86、HLA—DR、CD83HLA—DR并能刺激T细胞增殖与MLR（同基因、异基因)反应；T—DC具有抗原提呈功能、可诱导特异性杀伤。结论 用TNF-α预刺激4h的方法并在含有GM—CSF、IL-4的完全培养基中培养人外周血单核细胞8d可获得成熟的DC。

Tumor necrosis factor-alpha pretreatment for in vitro culture of mature dendritic cells]

OBJECTIVE: To explore a new method for in vitro culture of mature dendritic cells (DCs) by utilizing the maturation-inducing effect of tumor necrosis factor-alpha(TNF-alpha) on DC precursors. METHODS: Freshly isolated DC precursors (peripheral blood-derived monocytes) were initially cultivated for 4 h in RPMI 1640 supplemented with 10% fetal bovine serum and containing TNF-alpha (200 U/ml), followed by treatment with rhGM-CSF (800 U/ml) and interleukin (IL)-4 (500 U/ml) added into the medium. After 48 and 96 h, the 3 cytokines were simultaneously added to the medium for further culture. On day 8 of cell culture, the DCs were harvested and designated as T-DCs, which were subsequently subjected to examinations of flow cytometry analysis, assay by autologous and allogeneic mixed lymphocyte reaction (MLR) for estimating their maturation, and MTT assay for their specific killing activity. RESULTS: The T-DCs expressed specific surface markers (CD83,CD1a, CD54, CD11a, CD80, CD86, HLA-DR, CD83HLA-DR) of mature DCs, and stimulated strong T-cell proliferative responses in both autologous and autogeneic MLR. It also activated CD4+ T cells, which proliferated in response to autologous tumor, and specifically lysed tumor targets. CONCLUSION: The DCs thus cultured in vitro possess typical phenotype, and can present antigens to T cells, and this method provides an alternative in culturing mature and functionally competent DCs.