胰腺癌MiaPaCa-2细胞与树突细胞融合诱导肿瘤抗原特异性细胞毒T淋巴细胞能力的体外研究

In vitro study on the induction of antigen-specific cytotoxic T lymphocytes by fusion of dendritic cells with pancreatic canec r MiaPaCa-2 cells

目的：建立胰腺癌MiaPaCa-2细胞与树突细胞（ DC）融合的细胞，观察其体外诱导胰腺癌肿瘤抗原特异性细胞毒T淋巴细胞（ CTL）的能力。方法自胰腺癌患者外周血单核细胞中分离和培养DC，利用50％PEG-10％DMSO融合剂将MiaPaCa-2细胞融合到DC，以不加融合剂仅将DC与MiaPaCa-2共培养组及单纯DC组作为对照。采用FITC-CD86及PE-MUC1进行双标记，上流式细胞仪检测细胞融合率；MTT法检测各组DC存活率。按照DC与T淋巴细胞1∶10、1∶20、1∶40、1∶80的比例混合培养细胞，评价各组DC体外刺激自体T淋巴细胞的增殖能力；ELISA法检测各组DC体外激发的CTL的IL-2、IL-10、Granzyme B、IFN-γ分泌量。结果 DC与MiaPaCa-2融合细胞组的融合率为（42．30±7．30）％，明显高于共培养组的（7．21±1．06）％。 DC组、共培养组、融合细胞组DC的存活率分别为95．0％以上、85．0％、62．8％，融合细胞组 DC 存活率显著低于 DC 组及共培养组，差异有统计学意义（ P 值均＜0．05）。 DC∶T淋巴细胞为1∶10时，DC组、共培养组、融合细胞组DC刺激自体T细胞增殖指数分别为219±42、3584±317、8201±424，1∶20时分别为110±14、2179±104、6152±104，融合细胞组显著高于DC组及共培养组，差异均有统计学意义（ P值均＜0．05）；为1∶40、1∶80时3组细胞的T细胞增殖指数差异无统计学意义（ P值均＞0．05）。 DC∶T淋巴细胞为1∶10时，DC组、共培养组、融合细胞组DC激发的CTL的IL-2分泌量分别为（27．30±5．21）、（897．44±93．05）、（2243．80±381．46）ng／L；IL-10分泌量分别为（19．55±2．05）、（424．60±108．25）、（706．53±161．29） ng／L；Granzyme B 分泌量为（16．23±1．23）、（451．07±120．50）、（1327．77±205．15） ng／L；IFN-γ分泌量为（30．11±4．32）、（982．00±124．68）、（2421．04±488．50）ng／L。融合细胞组激发的CTL的细胞因子分泌量显著高于DC组及共培养组，差异均有统计学意义（P值均＜0．05）。结论 DC与MiaPaCa-2融合的细胞具备体外诱导胰腺癌肿瘤抗原特异性CTL的能力。

Objective To investigate the ability of inducing antigen-specific cytotoxic T lymphocytes （CTL） stimulated by dendritic cell （DC） fused with MiaPaCa-2 cells in vitro.Methods DC were isolated and cultured from peripheral blood mononuclear cells （PBMCs）.50%PEG and 10%DMSO were used to fuse MiaPaCa-2 cells and DC, and DC co-cultured with MiaPaCa-2 cells and DC alone served as control .The fusion efficiency was assessed by flow cytometry （ FCM） and 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 stimulated by DC in vitro was evaluated by mixed cell culture with DC in different ratios of 1∶10, 1∶20 and 1∶80.IL-2, IL-10, granzyme B and IFN-γreleased by antigen-specific CTLs were measured by ELISA assay.Results The fusion rate in DC fused with MiaPaCa-2 cells （ fused cells ） was （ 42 .30 ± 7.30）%, which was higher than （7.21 ±1.06）% in DC co-cultured with MiaPaCa-2 cells （ co-cultured cells）.The cell viability of DC, co-cultured cells and fused cells was ＆gt;95.0%, 85.0% and 62.8%, and fused cells had greatly lower cell viability than DC and co-cultured cells （P＆lt;0.05）.When DC and T cells were co-cultured at the ratio of 1∶10, T cell proliferation index in DC , co-cultured and fused cells was 219 ± 42, 3 584 ±317, 8 201 ±424, respectively.At the ratio of 1∶20, T cell proliferation index was 110 ±14, 2 179 ±104 , 6 152 ±104 .T cell proliferation index was higher in fused cells than that in DC and co-cultured cells （both P＆lt;0.05） at the ratio of 1∶10 and 1∶20, while the difference was not statistically significant at the ratio of 1∶40 and 1∶80 （P＆gt;0.05）.At the co-culture ratio of 1∶10, IL-2 secreted by CTL in DC, co-cultured and fused cells was （27.30 ±5.21 ）,（897.44 ±93.05）,（2 243.80 ±381.46） ng/L; IL-10 was （19.55 ± 2.05）, （ 424.60 ±108.25 ）, （ 706.53 ±161.29 ） ng/L; Granzyme B was （ 16.23 ±1.23 ）, （ 451.07 ± 120.50）,（1327.77 ±205.15）ng/L;IFN-γwas （30.11 ±4.32）、（982.00 ±124.68）、（2421.04 ±488.50） ng/L.Cytokines from the antigen-specific CTL induced by DC fused with MiaPaCa-2 cells were significantly higher than those by DC and DC co-cultured with MiaPaCa-2 cells （ all P＆lt;0.05）.Conclusions The fusion of DC and pancreatic cancer MiaPaCa-2 cells could stimulate tumor antigen-specific CTL in vitro .