胰腺癌Capan-2细胞与树突状细胞融合诱导特异性抗癌免疫应答的体外研究

Specific antitumor immune response induced by infusion of Capan-2 pancreatic cancer cells and dendritic cells: an in vitro study

目的观察人胰腺癌Capan-2细胞与树突状细胞(DC)融合后诱导的特异性抗肿瘤免疫反应。方法收集2013年4月-2015年3月于沈阳军区总医院消化科就诊的人类白细胞抗原(HLA)-A2^+胰腺癌患者6例,从患者外周血单个核细胞中分离并培养DC。所获DC分为3组:(1)使用聚乙二醇-二甲基亚砜诱导法,将DC与Capan-2细胞融合以负载肿瘤抗原;(2)DC与Capan-2细胞共培养;(3)单独DC培养。通过流式细胞术检测PE-MUC4/FITC-CD86抗体双标细胞评估融合率;应用四甲基偶氮唑盐法检测各组DC的存活率变化。使用干扰素(IFN)γ酶联免疫法检测DC诱导的细胞毒T淋巴细胞(CTL)活化反应。采用51Cr标准细胞毒实验检测DC诱导的抗原特异性CTL对体外胰腺癌细胞的杀伤作用。多组间比较采用方差分析,进一步两两比较采用LSD-t检验。结果DC与Capan-2融合细胞同时表达DC表型(CD86)和MUC4分子,CD86与MUC4双阳性表达率为(38.30±7.30)%,明显高于共培养组(7.21±1.06)%;融合细胞组DC存活率呈时间依赖性下降,转染后96 h的存活率降低至62.81%,而与Capan-2细胞共培养组DC存活率稳定在80%以上,两组间差异有统计学意义(P<0.05)。DC-Capan-2融合细胞诱导的CTL 24 h IFNγ释放量为(85.34±2.97)U/ml,DC、Capan-2共培养组DC诱导的IFNγ释放量为(19.07±4.25)U/ml,两转染组的差异有统计学意义(P<0.05)。DC-Capan-2融合细胞诱导的特异性CTL能够有效识别和杀伤HLA-A2^+/MUC4^+的Capan-2细胞及HLAA2^+/MUC4-的PANC-1细胞,但不能有效识别和杀伤HLA-A2-/MUC4-的Mia Pa Ca-2细胞和HLA-A2-/MUC4^+的As PC-1细胞。结论胰腺癌细胞与DC融合后可诱导出显著的CTL抗肿瘤免疫反应,此过程受主要组织相容性复合体Ⅰ类抗原呈递的限制。

Objective To investigate the specific antitumor immune response induced by the infusion of Capan- 2 pancreatic cancer cells and dendritic cells（ DC）. Methods DC were isolated from the peripheral blood mononuclear cells（ PBMC） derived from 6 patients with pancreatic cancer and cultured. The DC obtained were divided into three groups. In group 1,PEG- DMSO was used for induction,and DC and Capan- 2 cells were fused to bear tumor antigens. In group 2,DC were cultured with Capan- 2 cells. In group 3,DC were cultured alone. Flow cytometry was used to detect PE- MUC4 / FITC- CD86 double- labeled cells and assess the fusion rate,and MTT assay was used to determine the changes in viability of DCs in each group. IFNγ enzyme- linked immunosorbent assay was used to detect the activation reactions of cytotoxic T lymphocytes（ CTLs） induced by DCs. The 51 Cr standard cytotoxicity test was used to determine the killing effect of antigen- specific CTLs induced by DCs on in vitro pancreatic cancer cells. An analysis of variance was used for comparison between multiple groups. The LSD- t test was used for comparision between any two groups. Results The DC- Capan- 2 fused cells expressed DC phenotype（ CD86） and MUC4 molecules and had a significantly higher double- positive rate for CD86 and MUC4 than the co- cultured group（ 38. 30% ±7. 30% vs 7. 21% ± 1. 06%）. In the fusion group,the viability of DCs decreased in a time- dependent manner and reached62. 81% at 96 hours after transfection,while in the co- cultured group,the viability of DCs was maintained above 80%. The viability of DC showed a significant difference between these two groups（ P〈0. 05）. The release of IFNγ showed a significant difference between CTLs induced by DC- Capan- 2 fused cells and those induced by DCs in the co- cultured group（ 85. 34 ± 2. 97 U / ml vs 19. 07 ± 4. 25 U / ml,P〈0. 05）. The specific CTLs induced by DC- Capan- 2 fused cells could effectively identify identified and killed the HLA- A2^＋/ MUC4~＋Capan- 2 cells and the HLA- A2^＋/ MUC4-PANC- 1 cells,but did not effectively identify and kill the HLA- A2-/ MUC4-Mia Pa Ca- 2 cells and the HLA- A2-/ MUC4~＋As PC- 1 cells. Conclusion The fusion of pancreatic cells and DCs can induce a pronounced CTL anti- tumor immune response in CTLs,which is limited by which was restricted by MHC class I antigen presentedpresentation.