摘要目的 探讨胰腺癌细胞FOXP3表达对树突细胞( DCs )活化及其免疫功能的影响. 方法设计并合成靶向FOXP3的siRNA(siRNA-FOXP3)及阴性对照siRNA(siRNA-NC),转染胰腺癌PANC1细胞,ELISA法检测细胞培养上清IL-10和TGF-β1含量. 分别收集两组转染的胰腺癌细胞上清液,与粒细胞-单核细胞集落刺激因子( GM-CSF)及IL-4联合诱导DCs分化. 流式细胞仪检测经转染及未转染细胞培养上清液处理后DCs免疫表型CD86、CD80、HLA-DR等的变化,ELISA法检测上清中IL-12p70、IFN-γ含量. 将经上清液处理后的DCs与淋巴细胞共培养,CCK-8法检测各组DCs诱导的淋巴细胞增殖能力及活化的T细胞( CTLs)对胰腺癌细胞的杀伤性. 结果 与转染siRNA-NC的PANC1细胞比较,转染siRNA-FOXP3 的PANC1细胞IL-10、TGF-β1分泌量显著减少 [ (8.93 ±3.06)ng/L比(26.60 ± 5.57)ng/L,(2 544 ±78)ng/L比(2 856 ±92)ng/L],其细胞培养上清处理后DCs的CD86、HLA-DR阳性表达率显著增加[ (28.10 ±3.11)%比(13.90 ±0.42)%,(66.15 ±4.17)%比(43.15 ±3.32)% ],IL-12p70、IFN-γ分泌量显著增加[(52.75 ±7.89) ng/L比(26.14 ±4.50) ng/L,(898.43 ±88.82) ng/L比(412.76 ±24.68)ng/L],DCs 与淋巴细胞以1:5、1:10、1:20比例共培养后淋巴细胞的增殖显著加速[(95.27 ±3.80)%比(71.77 ±5.70)%,(78.97 ±5.73)%比(52.30 ±8.72)%,(57.60 ±4.36)%比(43.73 ±6.01)%],以1:20、1:40比例培养后活化的CTL对PANC1细胞的杀伤率显著提高[(28.44 ± 5.20)%比(8.82 ±2.29)%,(40.85 ±5.15)%比(17.38 ±4.86)%],两组间的差异均有统计学意义(P值均<0.05). 结论 胰腺癌细胞FOXP3表达显著抑制DCs的活化及其免疫功能.

abstractsObjective To investigate the influence of FOXP 3 expression in pancreatic carcinoma cells (PCCs) on the maturation and immunologic function of dendritic cells (DCs).Methods The siRNA sequences targeting FOXP3 gene (siRNA-FOXP3) and negative control siRNA (siRNA-NC) were specifically designed and transfected into PCCs , then the level of IL-10 and TGF-β1 of culture supernatant were detected by ELISA.The supernatants of pancreatic carcinoma cell transfected by FOXP 3-siRNA were collected , then it was mixed with GM-CSF and IL-4 to induce the differentiation of DCs .Flow cytometric analysis were used to measure the expression of surface markers CD 86 , CD80 , HLA DR on DCs which were treated with supernatants . The levels of IL-12p70, IFN-γin supernatants were detected by ELISA .The DCs were co-cultured with T lymphocytes, and then the lymphocytes proliferation and cytoxicity were analyzed by CCK -8 assays.Results Compared with PANC1 with siRNA-NC transfection, PANC1 with siRNA-FOXP3 transfection had a decreased expression of IL-10, TGF-β1 [(8.93 ±3.06)ng/L vs (26.60 ±5.57)ng/L;(2 544 ±78)ng/L vs (2 856 ± 92)ng/L], the positive expression rate of CD86, HLA DR in DCs cultured in the medium containing the supernatants of the pancreatic carcinoma cell transfected by siRNA-FOXP3 was significantly increased [(28.10 ±3.11)%vs (13.90 ±0.42)%;(66.15 ±4.17)%vs (43.15 ±3.32)%], the expression of IL-12p70, IFN-γwas significantly increased [(52.75 ±7.89)ng/L vs (26.14 ±4.50)ng/L, (898.43 ±88.82) ng/L vs (412.76 ±24.68) ng/L], after co-culture with lymphocytes at ratios of 1:5, 1:10, 1:20, the proliferation was significantly increased [(95.27 ±3.80)% vs (71.77 ±5.70)%, (78.97 ±5.73)% vs (52.30 ±8.72)%, (57.60 ±4.36)% vs (43.73 ±6.01)%], and the cytoxicity of CTL to PANC1 cells with 1:20, 1:40 co-culture was significantly increased [(28.44 ±5.20)% vs (8.82 ±2.29)%, (40.85 ± 5.15)% vs (17.38 ±4.86)%], and the difference between the two groups was statistically significant (P<0.05).Conclusions FOXP3 expression in PCCs can inhibit the maturation and immunologic function of DCs.