IL-10受体沉默的树突状细胞瘤苗对食管鳞癌细胞体内外免疫活性的影响  

Effects of IL⁃10 receptor⁃silenced DC vaccine on the immune activity of esophageal squamous cell carcinoma cells in vivo and in vitro

作　　者：朱晓磊 雷秀文 刘婷婷 朱自江 田拂晓 王栋 ZHU Xiaolei;LEI Xiuwen;LIU Tingting;ZHU Zijiang;TIAN Fuxiao;WANG Dong(Department of Cardiothoracic Oncology,Zhangye People′s Hospital Affiliated to Hexi University,Zhangye 734000,China;Operating Room,Zhangye People′s Hospital Affili-ated to Hexi University;Department of Chest Surgery,Central Hospital of Gansu Province;Department of Nutrition,Zhangye People′s Hospital Affiliated to Hexi University)

机构地区：[1]河西学院附属张掖人民医院心胸肿瘤外科,张掖734000 [2]河西学院附属张掖人民医院手术室 [3]甘肃省中心医院胸外科 [4]河西学院附属张掖人民医院营养科

出　　处：《山西医科大学学报》2024年第10期1251-1261,共11页Journal of Shanxi Medical University

基　　金：甘肃省科技计划项目(24JRRG005);甘肃省高等学校创新基金项目(2021B-234);张掖市市级科技计划课题(ZY2022BJ08);河西学院校长基金青年科研项目(QN2023009);国家自然科学基金资助项目(31760259)。

摘　　要：目的探讨IL-10受体(IL-10R)沉默的树突状细胞(DC)瘤苗对食管鳞癌细胞体内外免疫活性的影响。方法从食管鳞癌患者外周血中提取DC,使用倒置显微镜和扫描电镜对DC细胞形态进行鉴定,流式细胞仪上机检测DC细胞的表型。将TE11细胞分为空白对照组、DC-CTL组、NC-shRNA-DC-CTL组和IL-10R-shRNA-DC-CTL组。制作负载热休克凋亡的TE11细胞抗原的DC,然后使用Lipofectamine 3000分别进行IL-10R-shRNA慢病毒和NC-shRNA慢病毒转染,最后通过不同处理的DC诱导特异性细胞毒性T淋巴细胞(CTL)。通过MTT法检测DC对TE11细胞的杀伤率,采用ELISA法检测TE11细胞培养上清液中IL-12和干扰素(IFN)-γ含量。将荷瘤小鼠随机分为对照组、未转染的DC组(DC组)、NC-shRNA-DC组和IL-10R-shRNA-DC组,每组6只。将DC悬液或生理盐水注射至小鼠淋巴结,每周注射1次,共注射4次。每隔7 d用游标卡尺测量肿瘤体积。采用免疫组织化学染色检测肿瘤组织中Ki67表达水平,TUNEL染色检测肿瘤组织TUNEL阳性率,流式细胞术分析肿瘤浸润淋巴细胞中CD8^(+)T细胞的比例,采用ELISA法检测肿瘤组织中IL-12和IFN-γ水平,采用RT-qPCR检测肿瘤组织中IL-10 mRNA和IL-10R mRNA水平。采用Western blot检测肿瘤组织中IL-10、IL-10R、信号转导子和转录激活子3(STAT3)、p-STAT3、程序性细胞死亡蛋白1(PD-1)和程序性死亡配体1(PD-L1)的蛋白表达水平。结果倒置显微镜和扫描电镜证实分离的细胞符合DC形态学,DC的特异性标志物CD11c、MHC-Ⅱ、HLA-DR和CD83的阳性率分别为91.43%,89.53%,88.62%和90.74%。与空白对照组相比,DC-CTL组、NC-shRNA-DC-CTL组和IL-10R-shRNA-DC-CTL组TE11细胞杀伤率升高(P<0.05),TE11细胞培养上清液中IL-12和IFN-γ的水平升高(P<0.05);与DC-CTL组和NC-shRNA-DC-CTL组相比,IL-10R-shRNA-DC-CTL组TE11细胞杀伤率升高(P<0.05),TE11细胞培养上清液中IL-12和IFN-γ的水平升高(P<0.05)。与对照组相比,DC组、NC-shRNA-DC组和IL-10Objective To explore the effect of interleukin(IL)‐10 receptor(IL‐10R)‐silenced dendritic cell(DC)vaccine on the im‐mune activity of esophageal squamous cell carcinoma cells in vivo and in vitro.Methods DCs were extracted from the peripheral blood of esophageal squamous cell carcinoma patients,and then the morphology of DC cells was identified by inverted microscope and scanning electron microscope,and the phenotype of DC cells was detected by flow cytometry.TE11 cells were divided into blank con‐trol group,DC‐CTL group,NC‐shRNA‐DC‐CTL group and IL‐10R‐shRNA‐DC‐CTL group.DCs loaded with antigen of heat shock apoptotic TE11 cell were prepared,and then transfected with IL‐10R‐shRNA lentivirus and NC‐shRNA lentivirus using Lipofectamine 3000,respectively.DCs with different treatment were used to induce specific cytotoxic T lymphocytes(CTL).The killing rate of TE11 cells by DC was detected by MTT method,and the levels of IL‐12 and interferon(IFN)‐γin TE11 cell culture supernatant were detected by ELISA method.Tumor‐bearing mice were randomly divided into control group,DC group,NC‐shRNA‐DC group and IL‐10R‐shRNA‐DC group,with 6 mice in each group.DC suspension or physiological saline was injected into the lymph nodes of mice once a week for four times.Tumor volume was measured every seven days with a vernier caliper.Ki67 expression in tumor tissues was detected by immunohistochemical staining,and the positive rate of TUNEL in tumor tissues was detected by TUNEL staining.The dis‐tribution of CD8^(+)T cells in tumor‐infiltrating lymphocytes was analyzed by flow cytometry.The levels of IL‐12 and IFN‐γin tumor tissues were detected by ELISA method.The expression levels of IL‐10 mRNA and IL‐10R mRNA in tumor tissues were detected by RT‐qPCR.The protein expression levels of IL‐10,IL‐10R,signal transducer and activator of transcription 3(STAT3),p‐STAT3,pro‐grammed cell death protein 1(PD‐1)and programmed death ligand 1(PD‐L1)were detected by Western

关 键 词：食管鳞癌 树突状细胞瘤苗 白细胞介素-10受体 免疫 抗原呈递能力 信号转导子和转录激活子3 程序性细胞死亡蛋白1 

分 类 号：R735.1[医药卫生—肿瘤]