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

陈江; 许文达; 李宏宇; 赵家钧; 郭晓钟

doi:10.3969/j.issn.1001-5256.2016.05.013

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

DOI: 10.3969/j.issn.1001-5256.2016.05.013

沈阳军区总医院消化内科

基金项目:

国家自然科学基金资助项目(81071982)；

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中图分类号: R735.9
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出版历程
- 出版日期: 2016-05-20

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

Department of Gastroenterology, General Hospital of Shenyang Military Region

Research funding:

摘要

摘要:
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...
- 胰腺肿瘤 /
- 树突细胞 /
- T淋巴细胞,细胞毒性 /
- 细胞融合
Abstract:
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.
- pancreatic neoplasms /
- dendritic cells /
- T lymphocytes,cytotoxic /
- cell fusion

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参考文献(14)

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