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  

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

作　　者：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] Jiang Xiaofeng Yuan Xiaopeng Zhang Dawei Lu Haiwu Wen Zilong Zheng Qiang Liu Songhang Xue Ping(Department of Hepatobiliary Surgery, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China)

机构地区：[1]广州医科大学附属第二医院肝胆胰外科,510260

出　　处：《中华普通外科学文献（电子版）》2017年第4期217-221,共5页Chinese Archives of General Surgery(Electronic Edition)

基　　金：广东省社会发展领域科技计划项目(2014A020212511)

摘　　要：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DC CIK=1:10、1:20、1:40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20:1和40:1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10:1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。Objective To investigate the morphological changes and proliferation of cholangiocar-cinoma 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 1：10,1：20,1：40. 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 10：1. At the effective target ratio of 20：1 and 40：1, 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.

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 

分 类 号：R735.8[医药卫生—肿瘤]