胰腺癌细胞FOXP3表达对DCs活化及其免疫功能的影响

Effect of FOXP3 expression in pancreatic carcinoma cells on the maturation and immunologic function of dendritic cells

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、CD80HLA—DR等的变化，ELISA法检测上清中IL-12，pTO、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PANCl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，（25444-78）ng／L比（2856±92）ng／L]，其细胞培养上清处理后DCs的CDmHLA—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PANCl细胞的杀伤率显著提高[（28．44±5．20）％比（8．82±2．29）％，（40．854-5．15）％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。

Objective To investigate the influence of FOXP3 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 FOXP3-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 CD86 , CD80, HLA DR on DCs which were treated with supernatants. The levels of IL-12p70, IFN-γ in 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 euhured in the medium containing the supernatants of the pancreatic carcinoma cell transfeeted by siRNA-FOXP3 was significantly increased [ （28.10±3.11）% vs （13.90±0.42）%; （66.15±4.17）% vs （43.15±3.32）%1, the expression of IL- 12p70, IFN-γ was 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 eo-euhure with lymphoeytes 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 ） % I, and the eytoxieity of CTL to PANC1 cells with 1 ： 20, 1 ： 40 eo-euhure was significantly increased [ （ 28.44± 5.20） % vs （ 8.82 ±2.29 ） %, （40.85± 5.15）% vs （17.38 ± 4.86）% 1, 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.