摘要
目的比较人胰腺癌MiaPaCa-2细胞总RNA电转染树突细胞(Dendritic Cell,DC)与DCMiaPaCa-2融合细胞体外激发抗原特异性细胞毒T淋巴细胞(Cytotoxic T Lymphocyte,CTL)能力的差异。方法自6例胰腺癌患者外周血单核细胞中分离、培养DC。使用电穿孔法将MiaPaCa-2细胞总RNA转染DC,使用细胞融合方法将胰腺癌MiaPaCa-2细胞抗原负载DC,以未负载抗原的DC为对照。使用流式细胞术(FCM)检测PE-MUC/FITC-CD86抗体双标细胞评估融合效率;四甲基偶氮唑盐(MTT)检测转染各组DC存活率;混合细胞培养法评价各组DC体外刺激自体T淋巴细胞增殖能力;ELISA法检测各组DC体外激发抗原特异性CTL因子释放量。结果采用PEG-DMSO诱导的DC与MiaPaCa-2的融合细胞同时表达DC表型和MUC1分子,CD86与MUC1双阳性表达率为(42.3±7.30)%;融合细胞组DC存活率呈时间依赖性下降,转染后96h的存活率降低至62.81%,而MiaPaCa-2总RNA转染组DC细胞存活率稳定在85%左右,两组间差异有统计学意义(P<0.05);转染MiaPaCa-2总RNA DC刺激自体T细胞增殖指数(DC:T=1:10)为8432±611.25,显著高于DC-MiaPaCa-2融合细胞(DC:T=1:10)5672±107.51(P<0.05);且MiaPaCa-2总RNA转染DC激发特异性CTL分泌IL-12p70、IL-10和IFN-γ细胞水平亦显著异于DC-MiaPaCa-2融合细胞(P<0.05)。结论胰腺癌细胞总RNA转染DC较胰腺癌-树突融合细胞有更强的体外抗原特异性CTL激发能力。
Objective To compare the ability of specific cytotoxic T lymphocytes（CTL） induction stimulated by dendritic cells（DC） transfected with total RNA of human pancreatic cancer MiaPaCa-2 cell lines with DC fused with MiaPaCa-2 cell lines. Method DCs were isolated and cultured from peripheral blood mononuclear cells（PBMCs）. Total RNA derived from MiaPaCa-2 cell lines was transfected into DCs by electroporation. DCs were fused with MiaPaCa-2 cells and the fusion efficiency was assessed by flow cytometry（FCM）. DC-MiaPaCa-2 hybrids were identified as PE-MUC1/FITC-CD86 double positive cells. The survival rate of DC was determined by MTT method. The lymphocyte proliferation ability was evaluated by mixed cell culture method. The cytokines releasing of antigen-specific CTLs were measured by ELISA assay. Results DC-MiaPaCa-2 hybrids which possessed both the phenotype of DC and MUC1 protein. The co-expression rate of both CD86 and MUC1 positivity was（42.3 ± 7.30）%. The survival rate of DCs transected with MiaPa Ca-2 total RNA were stabilized around 85% and the survival rate of DCs fused with MiaPaCa-2 cells was62.81% at 96 hours（P〈0.05）. The autologous T cell proliferation index of the total RNA of MiaPaCa-2 celllines transfection DC group was 8432 ± 611.25, significantly higher than the DC-MiaPaCa-2 hybrids 5672 ±107.51（P〈0.05）; The secretion of IL-12p70, IL-10 and IFN-γ levels of the total RNA of MiaPaCa-2 cell lines transfection DC-specific CTL were significantly different with DC-MiaPaCa-2 hybrids（P〈0.05）.Conclusion DCs transfected with the total RNA of pancreatic cancer cells had a stronger ability to stimulate specific CTL in vitro than the DCs fused with pancreatic cancer cells.
出处
《现代消化及介入诊疗》
2016年第5期689-694,共6页
Modern Interventional Diagnosis and Treatment in Gastroenterology
基金
国家自然科学基金(81071982)
