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中华普通外科学文献(电子版)

中华普通外科学文献(电子版) ›› 2017, Vol. 11 ›› Issue (04) : 217 -221. doi: 10.3877/cma.j.issn.1674-0793.2017.04.001

所属专题： 文献；

论著

胆管癌全抗原致敏树突状细胞和细胞因子诱导杀伤细胞共培养体外抑瘤活性研究
蒋小峰1, 袁晓鹏1, 张大伟1, 卢海武1, 温子龙1, 郑强1, 刘颂航1, 薛平1,†()   
  1. 1. 510260　广州医科大学附属第二医院肝胆胰外科
  • 收稿日期:2017-06-12 出版日期:2017-08-01
  • 通信作者: 薛平
  • 基金资助:
    广东省社会发展领域科技计划项目(2014A020212511)

Study of dendritic cells co-cultured with cytokine induced killer cells sensitized by cholangiocarcinoma antigen on antitumor activity

Xiaofeng Jiang1, Xiaopeng Yuan1, Dawei Zhang1, Haiwu Lu1, Zilong Wen1, Qiang Zheng1, Songhang Liu1, Ping Xue1,†()   

  1. 1. Department of Hepatobiliary Surgery, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China
  • Received:2017-06-12 Published:2017-08-01
  • Corresponding author: Ping Xue
  • About author:
    Corresponding author: Xue Ping, Email:
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引用本文：

蒋小峰, 袁晓鹏, 张大伟, 卢海武, 温子龙, 郑强, 刘颂航, 薛平. 胆管癌全抗原致敏树突状细胞和细胞因子诱导杀伤细胞共培养体外抑瘤活性研究[J]. 中华普通外科学文献(电子版), 2017, 11(04): 217-221.

Xiaofeng Jiang, Xiaopeng Yuan, Dawei Zhang, Haiwu Lu, Zilong Wen, Qiang Zheng, Songhang Liu, Ping Xue. Study of dendritic cells co-cultured with cytokine induced killer cells sensitized by cholangiocarcinoma antigen on antitumor activity[J]. Chinese Archives of General Surgery(Electronic Edition), 2017, 11(04): 217-221.

目的

通过胆管癌全抗原致敏树突状细胞（DC）联合细胞因子诱导杀伤细胞（CIK），探讨DC-CIK细胞的形态改变及增殖情况，检测抗原致敏DC-CIK细胞的抗肿瘤活性。

方法

分离人外周血单个核细胞（PBMC）后取贴壁细胞作为前体DC，悬浮细胞用于CIK培养；经rhGM-CSF、rhIL-4、rhTNF-α诱导DC成熟；成熟DC中加入胆管癌细胞株RBE肿瘤抗原进行致敏，作为抗原致敏DC组；经rhIL-2、IL-1β诱导CIK成熟；按DCCIK=110、120、140的比例进行混合培养；流式细胞术检测DC表面标记（CD86、CD83、CD40、HLA-DR、CD1α、CD80）；流式细胞术检测CIK、未致敏DC-CIK、致敏DC-CIK共培养细胞表面标记（CD3、CD8、CD56）；采用萤火虫萤光素酶法检测CIK、未致敏DC-CIK、致敏DC-CIK抑瘤率；采用酶联免疫吸附测定（ELISA）法对细胞因子IL-12、IFN-γ的表达水平进行检测。

结果

DC-CIK细胞共培养，流式细胞学技术检测表型发现抗原致敏DC-CIK组的CD3+CD8+和CD3+CD56+细胞亚型表达均为最高，其次为未致敏DC-CIK组，最低为CIK组。效靶比为201和401时，致敏DC-CIK组的抑瘤率明显高于未致敏DC-CIK组和CIK组（P<0.05），而效靶比为101时，CIK组、未致敏DC-CIK组、致敏DC-CIK组的抑瘤率差异无统计学意义。抗原致敏DC较抗原未致敏DC及诱导前DC细胞状态好，生长增殖迅速。在抗原致敏后，致敏DC-CIK组的细胞因子IL-12、IFN-γ表达水平明显高于未致敏DC-CIK组和CIK组（P<0.05）。

结论

体外异体胆管癌全抗原可有效致敏健康人体DC细胞分化成熟，表型及增殖能力提高。使用体外致敏DC-CIK共培养作为效应细胞，具有较高的增殖率，在效靶比相同的情况下，抗原致敏DC-CIK对RBE有更强的杀伤性活性。

关键词: 胆管肿瘤, 树突细胞, 细胞因子诱导杀伤细胞, 肿瘤抑制
Objective

To investigate the morphological changes and proliferation of cholangiocarcinoma antigen sensitized DC-CIKs and to detect its antitumor activity.

Methods

DC and CIK were generated by culturing peripheral blood mononuclear cells (PBMCs) of healthy blood donors. Adherent cells were collected to generate DC, and non-adherent cells were collected to generate CIK. After being matured with rhGM-CSF, rhIL-4 and rhTNF-α, DC were sensitized with RBE tumor antigen lysates. CIK was matured with rhIL-2, IL-1β. DC and CIK were co-cultured at the ratio of 1:10, 120, 140. The DC mature phenotype were analyzed by flow cytometry. The anti-tumor effect of the co-culture sensitized DC and CIK was evaluated by luciferase. The secretion of IL-12, IFN-γ were assayed by ELISA.

Results

The growth and proliferation of antigen-sensitized DC was more potent than non- antigen-sensitized DC and pre-induced DC cells. The antitumor activity of the antigen-sensitized DC-CIK group was not significantly higher than that of the non-sensitized DC-CIK group at the effective target ratio of 101. At the effective target ratio of 201 and 401, the antitumor activity of antigen-sensitized DC-CIK group was significantly higher than that of non-sensitized DC-CIK group (P<0.05). The expression of cytokines (IL-12, IFN-γ) in antigen-sensitized DC-CIK group was significantly higher than that in non-sensitized DC-CIK group (P<0.05) and CIK group (P<0.05).

Conclusions

Allogeneic cholangiocarcinoma antigen can effectively sensitize DC cell of healthy human and promote proliferation ability, maturation and phenotype. The antigen-sensitized DC-CIK had the stronger cytotoxic activity against RBE than that of non-sensitized DC-CIK and CIK.

Key words: Bile duct neoplasms, Dendritic cells, Cytokine-induced killer cells, Tumor suppressor
图1 CIK表型流式细胞术检测散点图
表1 三组CIK表型检测结果（%）
组别 CD3+CD8+双阳性细胞率 CD3+CD56+双阳性细胞率
CIK组 49.49±3.79 14.35±2.46
未致敏DC-CIK组 73.66±3.62a 14.29±1.53
致敏DC-CIK组 85.41±2.61a 56.78±2.38a
统计值 16.27 13.82
P值 0.001 0.001
图2 RBE致敏树突状细胞(DC)和细胞因子诱导杀伤细胞(CIK)共培养的抑瘤作用，*P<0.05为差异有统计学意义
表2 三组与RBE靶细胞的抑瘤率检测（%）
组别 10∶1 20∶1 40∶1
CIK组 18.94±2.65 26.88±2.97 42.44±3.25
未致敏DC-CIK组 19.58±1.80 37.56±2.54 55.48±3.44
致敏DC-CIK组 25.28±2.08 55.50±3.65 70.88±7.36
统计值 6.64 7.82 9.21
P值 0.01 0.01 0.01
表3 三组细胞因子IL-12、IFN-γ的表达检测
组别 IL-12（μg/L） INF-γ（ng/L）
CIK组 13.92±10.24 207.82±62.78
未致敏DC-CIK组 74.35±37.26 503.42±108.26
致敏DC-CIK组 94.67±39.13 552.21±138.21
统计值 11.34 16.27
P值 0.01 0.001
[1]
Razumilava N, Gradilone SA, Smoot RL, et al. Non-canonical Hedgehog signaling contributes to chemotaxis in cholangiocarcinoma[J]. J Hepatol, 2014, 60(3):599-605.
[2]
El Ansary M, Mogawer S, Elhamid SA, et al. Immunotherapy by autologous dendritic cell vaccine in patients with advanced HCC[J]. J Cancer Res Clin Oncol, 2013, 139(1):39-48.
[3]
Kajihara M, Takakura K, Ohkusa T, et al. The impact of dendritic cell-tumor fusion cells on cancer vaccines past progress and future strategies[J]. Immunotherapy, 2015, 7(10):1111-1122.
[4]
Radford KJ, Tullett KM, Lahoud MH. Dendritic cells and cancer immunotherapy[J]. Curr Opin Immunol, 2014, 27:26-32.
[5]
Goc J, Germain C, Vo-Bourgais TK, et al. Dendritic cells in tumor-associated tertiary lymphoid structures signal a Th1 cytotoxic immune contexture and license the positive prognostic value of infiltrating CD8+ T cells[J]. Cancer Res, 2014, 74(3):705-715.
[6]
Zhen Z, Yang K, Ye L, et al. HLA-E inhibitor enhances the killing of neuroblastoma stem cells by co-cultured dendritic cells and cytokine-induced killer cells loaded with membrane-based microparticles[J]. Am J Cancer Res, 2017, 7(2):334-345.
[7]
Bol KF, Schreibelt G, Gerritsen WR, et al. Dendritic cell-based immunotherapy: state of the art and beyond[J]. Clin Cancer Res, 2016, 22(8):1897-906
[8]
Patel VI, Metcalf JP. Identification and characterization of human dendritic cell subsets in the steady state: a review of our current knowledge[J]. J Investig Med, 2016, 64(4):833-847.
[9]
Rutella S, Locatelli F. Is there a role for cytokine-induced killer cells in cancer immunotherapy?[J]. Immunotherapy, 2012, 4(9):867-869.
[10]
Pan Y, Wu Y, Ji J, et al. Effect of cytokine-induced killer cells on immune function in patients with lung cancer [J]. Oncol Lett, 2016, 11(4):2827-2834.
[11]
Zhao X, Ji CY, Liu GQ, et al. Immunomodulatory effect of DC/CIK combined with chemotherapy in multiple myeloma and the clinical efficacy[J]. Int J Clin Exp Pathol, 2015, 8(10):13146-13155.
[12]
Chen X, Du Y, Hu Q, et al. Tumor-derived CD4+CD25+regulatory T cells inhibit dendritic cells function by CTLA-4[J]. Pathol Res Pract, 2017, 213(3):245-249.
[13]
刘璐，李琳，伍衡, 等. 不同诱导方法对人外周血树突状细胞体外成熟的影响[J/CD]. 中华普通外科学文献(电子版), 2012, 6(1):45-50.
[14]
Wei H, Hongya P, Linlin J, et al. IFN-gamma enhances the anti-tumour immune response of dendritic cells against oral squamous cell carcinoma[J]. Arch Oral Biol, 2011, 56(9):891-898.
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