中国免疫学杂志2024，Vol.40Issue(6)：1189-1196,8.DOI:10.3969/j.issn.1000-484X.2024.06.013
CRISPR/CAS9敲除PD-1的肿瘤浸润T淋巴细胞回输对小鼠结肠癌的治疗作用
Therapeutic effect of reinfusion of tumor-infiltrating lymphocyte with CRISPR/CAS9 knockout PD-1 on colon cancer in mice
摘要
Abstract
Objective:To investigate therapeutic effect of reinfusion of tumor-infiltrating lymphocyte(TIL)with clustered regularly interspaced short palindromic repeats/CRISPR-associated 9(CRISPR/CAS9)knockout programmed death-1(PD-1)on colon cancer in mice.Methods:Subcutaneous injection of CT26 was used to establish mouse colon cancer model.TIL was extracted from tumor tissue of three model mice,and peripheral blood lymphocytes were extracted.PD-1 gene was knocked out of TIL.Reinfusion experiments were divided into control group(Control),lymphocyte group(Lym),tumor-bearing mouse TIL group(TIL),lentivirus empty empty group(pVSV-G-PX458-NC)and PX458-PD-1-sgRNA1 group(PD-1-sgRNA1),with 10 mice in each group.Tumor tissue quality and tumor inhibition rate were detected in each group.TUNEL was used to detect cell apoptosis in tumor tissues of mice.ELISA was used to detect contents of TNF-α and IFN-γ in tumor tissues of mice.Immunohistochemistry was used to detect expressions of CD4+T and CD8+T cells in tumor tissue.Immunofluorescence was used to detect expressions of proliferating cell nuclear antigen-67(Ki-67)and vascular endothelial growth factor(VEGF).Western blot was used to detect expressions of PD-1 and its ligand PD-L1 in tumor tissues.Results:PD-1-sgRNA1 could significantly inhibit growth of mouse tumor cells in vivo,inhibit expressions of Ki-67 and VEGF in tumor tissues,as well as expressions of PD-1 and PD-L1,elevate apoptosis rate,contents of TNF-α and IFN-γ in tumor tissues,and expressions of CD4+T and CD8+T cells(all P<0.05).Conclusion:Reinfusion of TIL with CRISPR/CAS9 knockout PD-1 can significantly inhibit expressions of Ki-67 and VEGF in colon cancer mice,enhance infiltration of CD4+T and CD8+T cells,induce tumor cell apoptosis and inhibit tumor growth.关键词
CRISPR/Cas9/PD-1/结肠癌/肿瘤浸润淋巴细胞/肿瘤生长Key words
CRISPR/Cas9/PD-1/Colon cancer/Tumor-infiltrating lymphocyte/Tumor growth分类
医药卫生引用本文复制引用
瞿紫微,李晓辉,郭建辉,陈华涛,吴彪,孟庆彬..CRISPR/CAS9敲除PD-1的肿瘤浸润T淋巴细胞回输对小鼠结肠癌的治疗作用[J].中国免疫学杂志,2024,40(6):1189-1196,8.基金项目
武汉市卫健委医学科研项目(WX21A11). （WX21A11）
