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首页> 外文期刊>World Journal of Gastroenterology >A novel, rapid strategy to form dendritomas from human dendritic cells and hepatocellular carcinoma cell line HCCLM3 cells using mature dendritic cells derived from human peripheral blood CD14+ monocytes within 48 hours of in vitro culture
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A novel, rapid strategy to form dendritomas from human dendritic cells and hepatocellular carcinoma cell line HCCLM3 cells using mature dendritic cells derived from human peripheral blood CD14+ monocytes within 48 hours of in vitro culture

机译：一种新颖，快速的策略，在体外培养的48小时内，使用衍生自人外周血CD14 +单核细胞的成熟树突状细胞，从人树突状细胞和肝癌细胞系HCCLM3细胞形成树突状瘤

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AIM: Dendritomas formed by fusing cancer cells to dendritic cells have already been applied to clinical treatment trial of several types of cancers. Dendritic cells for the fusion in most trials and experiments were from blood monocytes in standard 7-d protocol culture, which requires 5-7 d of culture with granulocyte-macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), followed by 2-3 d of activation with a combination of proinflammatory mediators such as tumor necrosis factorα (TNFα), interleukin-1β (IL-1β), interleukin-6 (IL-6) and prostaglandin E_2(PGE_2). One study showed that mature monocyte-derived dendritic cells could be obtained within 48 h of in vitro culture with the same protocol as standard 7-d culture and referred to as FastDCs. Here we aimed to fuse human hepatocellular carcinoma cell line HCCLM3 cells with mature monocyte-derived dendritic cells within 48 h of in vitro culture (FastDC). METHODS: HCCLM3 cells were cultured in RPMI1640 with 150 mL/L fetal calf serum (FCS). CD14+monocytes from healthy human peripheral blood were purified with MACS CD14 isolation kit and cultured in six-well plates in fresh complete DC medium containing RPMI-1640, 20 mL/L heat inactivated human AB serum, 2 mmol/L L-glutamine, 100 μg/mL gentamicin, 1 000 U/mL GM-CSF and 500 U/mL IL-4 for 24 h, then proinflammatory mediators such as TNFα (1 000 U/mL), IL-1β (10 ng/mL), IL-6 (10 ng/mL) and PGE_2 (1 ng/mL) were supplemented for another 24 h, and thus mature FastDCs were generated. HCCLM3 cells and FastDCs were labeled with red fluorescent dye PKH26-GL and green fluorescent dye PKH67-GL respectively. After the red fluorescent-stained HCCLM3 cells were irradiated with 50 Gy, FastDCs and irradiated HCCLM3 cells were fused in 500 mL/L polyethylene glycol(PEG)+100 mL/L dimethyl sulfoxide (DMSO) to generate novel dendritomas. The FastDCs and novel dendritomas were immunostained with anti-CD80, anti-CD86, anti-CD83, anti-HLA-DR mAbs and analyzed by fluorescence-activated cell sorting (FACS). Novel dendritomas were nucleus-stained with Hoechst 33258 and analyzed by confocal laser scanning microscopy. RESULTS: Mature FastDCs with highly expressed surface markers CD80, CD86, CD83 and HLA-DR were generated within 48 h in vitro. Novel dendritomas with dual red-green fluorescence were constructed fast and successfully, and FACS analysis showed that the fusion efficiency was 24.27% and the novel dendritomas expressed the same activation markers as FastDCs. Confocal laser scanning microscopy analysis showed representative images of dendritomas. CONCLUSION: Dendritomas can be formed fast with mature FastDCs from healthy human peripheral blood monocytes (PBMC) by incubation with GM-CSF and IL-4 for 24 h and by activation with proinflammatory mediators for an additional period of 24 h. Owing to shorter time required for in vitro DCs development, the generation of these novel dendritomas reduced labor and cost. This rapid method for formation of dendritomas may represent a new strategy for immunotherapy of hepatocellular carcinoma.
机译：目的：通过将癌细胞与树突状细胞融合而形成的树突状瘤已被用于多种类型癌症的临床治疗试验。在大多数试验和实验中，用于融合的树突状细胞来自标准7-d方案培养中的血液单核细胞，需要与粒细胞-巨噬细胞-集落刺激因子（GM-CSF）和白介素-4（IL）一起培养5-7 d -4），然后结合促炎介质（如肿瘤坏死因子α（TNFα），白介素-1β（IL-1β），白介素-6（IL-6）和前列腺素E_2（PGE_2））激活2-3天。一项研究表明，成熟的单核细胞来源的树突状细胞可以在体外培养的48小时内以与标准7-d培养物相同的方案获得，并称为FastDCs。在这里，我们的目标是在体外培养（FastDC）的48小时内将人肝细胞癌HCCLM3细胞与成熟的单核细胞衍生的树突状细胞融合。方法：用150mL / L胎牛血清（FCS）在RPMI1640中培养HCCLM3细胞。使用MACS CD14分离试剂盒纯化健康人外周血中的CD14 +单核细胞，并在六孔板中于含有RPMI-1640、20 mL / L热灭活人AB血清，2 mmol / L L-谷氨酰胺， 100μg/ mL庆大霉素，1000 U / mL GM-CSF和500 U / mL IL-4持续24小时，然后是促炎性介质，例如TNFα（1000 U / mL），IL-1β（10 ng / mL），将IL-6（10 ng / mL）和PGE_2（1 ng / mL）补充24小时，从而生成成熟的FastDC。 HCCLM3细胞和FastDCs分别用红色荧光染料PKH26-GL和绿色荧光染料PKH67-GL标记。用50 Gy辐照红色荧光染色的HCCLM3细胞后，将FastDC和辐照过的HCCLM3细胞融合在500 mL / L聚乙二醇（PEG）+100 mL / L二甲基亚砜（DMSO）中，以生成新的树突状瘤。 FastDC和新型树突状瘤用抗CD80，抗CD86，抗CD83，抗HLA-DR mAb免疫染色，并通过荧光激活细胞分选（FACS）进行分析。新型树突状瘤用Hoechst 33258核染色，并通过共聚焦激光扫描显微镜进行分析。结果：成熟的FastDCs具有高表达的表面标记CD80，CD86，CD83和HLA-DR可在体外48小时内产生。快速，成功地构建了具有双红绿色荧光的新型树突状瘤，FACS分析表明融合效率为24.27％，新型树突状瘤表达的活化标记与FastDCs相同。共聚焦激光扫描显微镜分析显示树突状瘤的代表性图像。结论：通过与GM-CSF和IL-4孵育24 h，并与促炎介质激活另外24 h，可以使用来自健康人外周血单核细胞（PBMC）的成熟FastDC快速形成树突状瘤。由于体外DCs开发所需的时间较短，这些新型树突状瘤的产生减少了劳力和成本。这种快速形成树突状瘤的方法可能代表了肝细胞癌免疫治疗的新策略。

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