基于干扰素基因刺激因子的特异性靶向Dickkopf相关蛋白1的肿瘤疫苗对多发性骨髓瘤的抗肿瘤免疫应答  

Antitumor immune response of stimulator of interferon genes-based Dickkopf-related protein 1-targeted vaccine in multiple myeloma

作　　者：肖蓬莉 郭淑利 王慧睿 毛慧云 安万花 XIAO Pengli;GUO Shuli;WANG Huirui;MAO Huiyun;AN Wanhua(Department of Hematology,Luoyang Central Hospital Affiliated to Zhengzhou University,Luoyang 471009,Henan Province,China)

机构地区：[1]郑州大学附属洛阳中心医院血液科,河南洛阳471009

出　　处：《新乡医学院学报》2024年第10期911-918,共8页Journal of Xinxiang Medical University

基　　金：河南省科技攻关计划项目(编号:212102310751);中原学者工作站项目(编号:214400510018)。

摘　　要：目的探讨干扰素基因刺激因子(STING)激动剂ADU-S100是否可增强壳聚糖(CS)纳米颗粒介导的包含肿瘤特异性抗原Dickkopf相关蛋白1(DKK1)的DNA疫苗对多发性骨髓瘤(MM)的抗肿瘤免疫应答。方法应用复合物共沉淀法制备CS-DNA纳米颗粒。采用Zetasizer Nano-ZS激光粒度分析仪检测CS-DNA纳米颗粒的粒度和Zeta电位,分别通过凝胶阻滞分析和Western blot评估CS-DNA纳米颗粒的DNA保护效果和体内DNA表达效率。利用表达人DKK1(hDKK1)基因的慢病毒建立稳定表达hDKK1的MPC-11细胞(MPC-11-hDKK1),并将MPC-11-hDKK1细胞皮下接种于小鼠建立肿瘤模型。将荷瘤小鼠随机分为对照组(肌内注射CS-pcDNA3.1)、ADU-S100免疫组(皮下注射ADU-S100)、CS-pDKK1免疫组(肌内注射CS-pDKK1)和ADU-S100/CS-pDKK1联合免疫组(肌内注射CS-pDKK1+皮下注射ADU-S100),每组5只。各组荷瘤小鼠按照相应的免疫方案以10 d为间隔免疫3次。每周测量肿瘤大小。肿瘤细胞MPC-11-hDKK1接种后第42天,测量各免疫组小鼠的肿瘤重量;流式细胞术检测各免疫组小鼠脾脏中CD11c^(+)树突状细胞(DC)、CD8^(+)CD11c^(+)DC和主要组织相容性复合物Ⅱ类(MHCⅡ)+CD11c^(+)DC亚群的占比。采用重组hDKK1蛋白体外刺激各组小鼠的脾细胞,应用流式细胞术检测各免疫组CD8^(+)T淋巴细胞中EdU+细胞占比,并应用乳酸脱氢酶(LDH)细胞毒性检测试剂盒测定各组细胞毒性T淋巴细胞(CTL)的杀伤效应。结果CS-DNA纳米颗粒的粒径和Zeta电位分别约为(204.3±2.31)nm和(15.47±1.01)mV。凝胶阻滞分析表明,CS纳米颗粒包裹的DNA可被完全阻滞。Western blot分析表明,CS-DNA纳米颗粒可在体内有效表达。MPC-11-hDKK1细胞中DKK1蛋白的相对表达量显著高于MPC-11-Ctrl细胞(P<0.05)。肿瘤细胞MPC-11-hDKK1接种后第7、14天,ADU-S100免疫组、CS-pDKK1免疫组和ADU-S100/CS-pDKK1联合免疫组与对照组小鼠的肿瘤体积比较差异无统计学意义(P>0.05);接种后第21、28、35�Objective To explore whether stimulator of interferon genes(STING)agonist ADU-S100 could enhance the antitumor immune response of a chitosan(CS)nanoparticle-mediated DNA vaccine containing a tumor-specific antigen Dickkopf-related protein 1(DKK1)in multiple myeloma(MM).Methods CS-DNA nanoparticles were prepared by using the compound coprecipitation method.The particle sizes and Zeta potential of the CS-DNA nanoparticles were measured by using the Zetasizer Nano-ZS laser particle size analyzer.The DNA protection effect and in vivo DNA expression efficiency of the CS-DNA nanoparticles were assessed by using gel retardation assay and Western blot,respectively.The lentiviruses expressing human DKK1(hDKK1)genes were used to establish MPC-11 cells(MPC-11-hDKK1)which stably expressed hDKK1,and the MPC-11-hDKK1 cells were subcutaneously given to mice to construct tumor models.The tumor-bearing mice were randomly divided into a control group(intramuscular injection of CS-pcDNA3.1),an ADU-S100 immunization group(subcutaneous injection of ADU-S100),a CS-pDKK1 immunization group(intramuscular injection of CS-pDKK1)and an ADU-S100/CS-pDKK1 co-immunization group(intramuscular injection of CS-pDKK1+subcutaneous injection of ADU-S100),with 5 mice in each group.The tumor-bearing mice in each group were immunized 3 times at 10-day intervals according to the corresponding immunization schedule.The size of tumor was measured every week.On day 42 after MPC-11-hDKK1 cell inoculation,the tumor weight of mice in each immunization group was measured;the percentages of CD11c^(+)dendritic cell(DC),CD8^(+)CD11c^(+)DC and major histocompatibility complex class II(MHCII)+CD11c^(+)DC subsets in the spleen of mice in each immunization group were detected by using flow cytometry.The splenocytes of mice in each group were stimulated with recombinant hDKK-1 protein in vitro,the percentage of EdU+cells in CD8^(+)T lymphocytes in each immunization group was detected by using flow cytometry,and the killing effect of cytotoxic T lymphocyte(CTL)in each

关 键 词：干扰素基因刺激因子激动剂 壳聚糖纳米颗粒 DNA疫苗 抗肿瘤免疫 多发性骨髓瘤 

分 类 号：R733.3[医药卫生—肿瘤]