树突状细胞的扩增及培养上清液对胰腺癌细胞凋亡的研究

Amplification of DC and effect of culture supernatant on PC3 Apoptosis

目的体外观察胰腺癌(PC)患者树突状细胞(DC)增生成熟,DC分泌的细胞因子及DC诱导免疫效应细胞分泌的细胞因子对胰腺癌细胞PC3的凋亡作用.方法用淋巴细胞分离液分离获取外周血单核细胞(PBMC),贴壁法获取DC和去DC的单核细胞(即免疫效应细胞),分别用粒/巨噬细胞集落刺激因子(GM-CSF)1 000 U/ml,人白细胞介素-4(IL-4)500 U/ml,肿瘤坏死因子(TNF)-α 500 U/ml、PC3肿瘤相关(TAA)和人IL-2 100 U/ml培养DC和免疫效应细胞,观察DC生长状况,检测DC表型(CD1a、CD80、CD83、CD86)及DC培养上清液对PC3细胞的凋亡作用.结果体外多种细胞因子和肿瘤相关抗原能有效引起胰腺癌患者DC增殖0.5×105～1.0×105个/ml,高表达CD80、CD83、CD86.DC培养上清液和DC混合免疫效应细胞培养上清液均能有效地引起PC3凋亡.结论DC在胰腺癌免疫治疗中有重要作用.

Objective To observe dendritic cell (DC) proliferation and maturation, and the effects of cytokines secreted by DC and the cytokines by DC-induced immunoeffect cells on PC3 apoptosis ex vi- vo. Methods The DC and mononuclear cells without DC (namely immunoeffect cells) were isolated and purified from peripheral blood mononuclear cells (PBMC) in the patients with pancreatic cancer. The DC were cultured in the presence of granulocyte/ macrophage colony stimulating factor (1 000 U/ml), inter- leukin-4 (500 U/ml), tumor necrosis factor-α(500 U/ml) and extracted tumor-associated antigen (TAA) from human pancreatic cancer cell line PC3, and the immunoeffect cells were culture with inter- leukin-2 (100 U/ml), respectively. 5 or 6 days later, DC and immunoeffect cells were cocultured for two days ex vivo. The effects of DC phenotypes (CD1a, CD80, CD83 and CD86) and the culture supernatant of DC on the apoptosis of PC3 were determined. Results In vitro cytokines and tumor related antigens could effectively result in the DC amplification to 0. 5×10~5 ～1. 0×10~5 DC/ml and high expression of CD80, CD83 and CD86. Both the culture supernanant of DC and that of DC in combination with immu- noeffect cells could induce the apoptosis of PC3. conclusion DC might play an important role in the im- munotherapy of pancreatic cancer.