山西医科大学学报2024，Vol.55Issue(10)：1251-1261,11.DOI:10.13753/j.issn.1007-6611.2024.10.001
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
摘要
Abstract
Objective 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)-γ in 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-γ in 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 blot.Results Inverted microscopy and scanning electron microscopy confirmed that the isolated cells were consistent with DC morphology.The positive rates of specific markers CD11c,MHC-Ⅱ,HLA-DR and CD83 in DCs were 91.43%,89.53%,88.62%and 90.74%respectively.Com-pared with blank group,the TE11 cell killing rate was increased in DC-CTL group,NC-shRNA-DC-CTL group and IL-10R-shRNA-DC-CTL group(P<0.05),and the levels of IL-12 and IFN-γ in TE11 cell culture supernatants were increased(P<0.05).Compared with DC-CTL group and NC-shRNA-DC-CTL group,the TE11 cell killing rate was increased in IL-10R-shRNA-DC-CTL group(P<0.05),and the levels of IL-12 and IFN-γ in TE11 cell culture supernatants were increased(P<0.05).Compared with control group,the tumor vo-lume and the Ki67 positive rate in tumor tissues were decreased in DC group,NC-shRNA-DC group and IL-10R-shRNA-DC group(P<0.05),the TUNEL positive rate was increased(P<0.05),the proportion of CD8+T cells in tumor infiltrating lymphocytes and the levels of IL-12 and IFN-γ in tumor tissues were increased(P<0.05),and the relative levels of IL-10 and IL-10R mRNA and protein,the relative levels of STAT3 phosphorylation,and the relative levels of PD-1 and PD-L1 proteins in tumor tissues were decreased(P<0.05).Compared with DC group and NC-shRNA-DC group,the tumor volume and the Ki67 positive rate in tumor tissues were decreased in IL-10R-shRNA-DC group(P<0.05),the TUNEL positive rate was increased(P<0.05),the proportion of CD8+T cells in tumor infiltrating lymphocytes and the levels of IL-12 and IFN-γ in tumor tissues were increased,and the relative levels of IL-10 and IL-10R mRNA and protein,the relative levels of STAT3 phosphorylation,and the relative levels of PD-1 and PD-L1 proteins in tumor tissues were decreased(P<0.05).Conclusion IL-10R-silenced DC tumor vaccine has anti-esophageal squamous cell carcinoma activity in vivo and in vitro,and can effectively enhance the immune activity of CD8+T cells,and inhibit the activation of STAT3 and PD-1/PD-L1 immune checkpoint.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1Key words
esophageal squamous cell carcinoma/dendritic cell vaccine/interleukin-10 receptor/immunity/antigen pre-sentation ability/signal transducer and activator of transcription 3/programmed death-1分类
医药卫生引用本文复制引用
朱晓磊,雷秀文,刘婷婷,朱自江,田拂晓,王栋..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[J].山西医科大学学报,2024,55(10):1251-1261,11.基金项目
甘肃省科技计划项目(24JRRG005) （24JRRG005）
甘肃省高等学校创新基金项目(2021B-234) （2021B-234）
张掖市市级科技计划课题(ZY2022BJ08) （ZY2022BJ08）
河西学院校长基金青年科研项目(QN2023009) （QN2023009）
国家自然科学基金资助项目(31760259) （31760259）
