肺癌胸腔积液树突状细胞培养与鉴别及其抗肿瘤作用的观察

Generation and anti-tumor effect of dendritic cells from malignant pleural effusion of patients with lung cancer

目的:证实肺癌胸腔积液中存在树突状细胞(DCs)的前体细胞,体外培养可以获得成熟的DCs,并探讨DCs对自体癌细胞的杀伤作用。方法:通过Ficoll淋巴细胞分离液对15例肺癌患者的恶性胸腔积液进行密度梯度离心,以两步贴壁法分别获得DCs的前体细胞和自体癌细胞。前者加入粒细胞/巨噬细胞集落刺激因子(rhGM-CSF)、白细胞介素-4(rhIL-4)及肿瘤坏死因子-α(TNF-α)培养。显微镜观察细胞形态,流式细胞仪鉴定DCs表型。MTT法检测DCs对自体癌细胞的杀伤作用。结果:体外培养9d后获得大量成熟的DCs,具有典型树突状形态,且高表达HLA-DR〔(84.2±6.3)%〕、CD80〔(75.8±8.8)%〕、CD83〔(58.0±9.4)%〕和CD86〔(83.3±7.5)%〕;MTT法结果显示,DCs对自体癌细胞有较强的杀伤作用,当效靶比为20∶1时杀伤率为(50.62±7.51)%。结论:肺癌恶性胸腔积液中的DCs前体细胞经诱导培养后可转化为功能健全的成熟DCs,成熟的DCs对自体癌细胞有直接杀伤作用,为恶性胸腔积液的免疫治疗提供理论依据。

OBJECTIVE ： To clarify that there are progenitor cells of the dendritic cells （DCs） in malignant pleural effusions of patients with lung cancer, and test that, the matured DCs can be generated from the progenitor cells as well as to investigate the anti-tumor effect of DCs against autologous lung cancer cells in vitro. METHODS： Progenitor cells and au- tologous cancer cells were seperated by two-step adherence after Ficoll density gradient centrifugation from MPE in 15 patients with lung cancer. Progenitor cells were induced in vitro by human recombinant granulocyte macrophage colony stimulating factor （rhGM-CSF）,human recombinant interleukin-4 （rhIL-4） and tumor necrosis factor-α （TNF-α）. Morphology of DCs was observed by microscope. The expressions of HLA-DR,CD80,CD83 and CD86 on DCs were analyzed by flow- cytometry. The anti-tumor effect of DCs was evaluated by MTT assay. RESULTS.. A large number of morphologically typical dendritic cells were observed after the culturing for 9 days with high expression of HLA-DR[（84.3±6.3）%] ,CD80 [（75.8±8.8） %] ,CD83[（58. 0±9. 4） %] and CD86[（83.2±7.5） %]. DCs had a stronger killing role to the autologous lung cancer cells. When effector-target ration was 20 ： 1,the killing rate was （50.62 ± 7.51）%. CONCLUSIONS： Mature DCs can be induced from MPE in patients with lung cancer, which have tipical morphology and phenotypic. The matured DCs have anti-tumor effect against autologous lung cancer cells. These provid the theory support to the immune therapy of MPE.