肿瘤抗原负载树突状细胞联合CIK通过促进ASK1活化增强对人食管癌细胞的杀伤活性  

Tumorantigen-loaded dendritic cells combinedd with cytokine-inducedkiller(CIK)enhance the killing activity of human esophageal cancer cells by promoting ASK1 activation

作　　者：段铮[1] 李洪霖 胡斌 李云 黄莉[1] DUAN Zheng;LI Honglin;HU Bin;LI Yun;HUANG Li(The Second District of Tumor,Henan Province Hospital of TCM,Zhengzhou 450000,China)

机构地区：[1]河南省中医院肿瘤二区,河南郑州450000

出　　处：《细胞与分子免疫学杂志》2023年第6期501-508,共8页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金青年科学基金(81804057)。

摘　　要：目的探究肿瘤抗原负载的树突状细胞(Ag-DC)联合细胞因子诱导的杀伤细胞(CIK)对食管癌肿瘤细胞杀伤的影响及作用机制。方法诱导培养外周血DC和CIK,用Ag负载DC获得Ag-DC,将Ag-DC与CIK共培养。实验分为CIK组、DC联合CIK组、Ag-DC联合CIK组。流式细胞术检测细胞表型,噻唑蓝(MTT)法测定细胞对EC9706食管癌细胞的杀伤活性,异硫氰酸荧光素标记的膜联素V/碘化丙啶(annexin V-FITC/PI)双染法检测细胞凋亡率,免疫荧光染色检测磷酸化的细胞凋亡信号调节激酶1(p-ASK1)表达,Western blot法测定ASK1途径相关蛋白水平。构建食管癌移植瘤裸鼠模型,分为对照组、DC联合CIK组和Ag-DC联合CIK组。通过尾静脉注射对应免疫细胞进行治疗,每隔2 d测量一次肿瘤体积。21 d后处死所有裸鼠,取出瘤体,HE染色观察肿瘤组织病变情况,免疫组织化学染色法检测抗原KI-67(ki67)及ASK1表达。结果与单独CIK组和DC联合CIK组相比,Ag-DC与CIK共培养后,细胞中CD3+CD8+与CD3+CD56+的比例明显升高,对EC9706细胞的杀伤率明显增加,增加EC9706细胞凋亡,并促进ASK1的活化水平;与单独CIK组和DC联合CIK组比较,Ag-DC联合CIK处理的裸鼠移植瘤生长受到明显的抑制,21 d后观察到该组肿瘤组织块较小,肿瘤组织内细胞排列较为稀疏,肿瘤组织内ki67阳性率明显减少,而ASK1阳性率则明显增高。结论Ag-DC与CIK共培养后,能够明显提高对食管癌肿瘤细胞的杀伤活性,该作用机制可能与ASK1途径的激活相关。Objective To clarify the effect and mechanism of tumor antigen-loaded dendritic cells(Ag-DCs)combined with cytokine-induced killers(CIKs)on the kiling of esophageal cancer tumor cells.Methods Peripheral blood DCs and CIKs were induced and cultured,and the DCs were loaded with tumor antigen to obtain Ag-DCs,and Ag-DCs were co-cultured with CIKs.The experiment was divided into CIK group,DC combined with CIK group,Ag-DC combined with CIK group.Flow cytometry was used to detect the phenotype of cells.MTT assay was employed to determine the kiling activity against EC9706 cells.Annexin V-FITC/PI double staining was used to detect the apoptosis rate of cells,immunofluorescence staining to detect the expression of phosphorylated apoptotic signal-regulated kinase 1(p-ASK1)and Western blot analysis to detect the expression of AsK1 pathway related proteins.A nude mouse model of esophageal cancer transplantation tumor was constructed and divided into control group,DC combined with CIK group and Ag-DC combined with CIK group.The corresponding immune cells were injected into the tail vein for treatment and the tumor volume was measured every 2 days.After 21 days,all nude mice were sacrificed with the tumors taken out.HE staining was used to observe the tumor pathological changes and immunohistochemical staining was performed to detect the expression of ki67 and ASK1 in the tumortissue.Results Comparedwith the CIK group alone and the DC combined with CIK group,the ratio of CD3^(+)CD8^(+)and CD3 CD56^(+)in the cells significantly increased after Ag-DCs and CIKs co-culture,along with the increased kiling rate of EC9706 cells,increased apoptosis rate of EC9706 cells,and the improved activation level of ASK1.Compared with the CIK group and the DC combined with CIK group,the growth of the transplanted tumor in nude mice treated with Ag-DCs combined with ClKs was significantly inhibited,and after 21 days,it was observed that the tumor tissue mass in this group was relatively smaller,with sparsely arranged cells in the tumor tissue an

关 键 词：食管癌 肿瘤抗原 树突状细胞 细胞因子诱导的杀伤细胞 杀伤作用 

分 类 号：R392.12[医药卫生—免疫学] R735.1[医药卫生—基础医学] R735R730.51