安徽医科大学学报2024，Vol.59Issue(12)：2103-2111,9.DOI:10.19405/j.cnki.issn1000-1492.2024.12.006
基于AAV6-CRISPR-Cas9系统抑制NBR1表达对肺癌小鼠肿瘤免疫调控的影响
Inhibiting NBR1 expression using the AAV6-CRISPR-Cas9 system affects tumor immune regulation in lung cancer mice
摘要
Abstract
Objective To establish a NBR1-knockout lung cancer mouse model through CRISPR-Cas9 technology by using adeno-associated virus(AAV)as a vector to specifically inhibit NBR1 expression and to investigate the impact of NBR1 knockout on tumor growth and immune cell infiltration and regulation.Methods sgRNAs targeting mouse NBR1(Gene ID:17966)was designed using the online tool CRISPOR(http://crispor.tefor.net/crispor.py).AAV6 was utilized as the vector for sgRNA delivery,and the efficiency of gene knockout was confirmed using PCR and DNA sequencing methods.To determine the best AAV infection approach in mice,6 C57BL/6J mice were randomly divided into intranasal and endotracheal groups.After 28 days,lung tissue sections were assessed for enhanced green fluorescent protein expression to identify the more efficient infection method for subsequent ex-periments.Lung tumor growth,as well as immune cell infiltration and activation status in tumor tissues,were de-tected using methods including HE staining,immunohistochemistry,immunofluorescence,and flow cytometry.Re-sults DNA sequencing and immunofluorescence results indicated successful construction of the AAV6-U6-sgN-BR1-CAG-Cre-GFP vector with stable knockout efficiency.Fluorescence microscopy showed higher efficiency of lung infection in mice through intratracheal administration(P＜0.05).HE staining revealed reduced tumor area in mouse lungs after targeted NBR1 knockout compared to the control group(P＜0.01).Immunofluorescence and flow cytometry results demonstrated enhanced functional activity of CD8+T lymphocytes in lung cancer tissues of mice with targeted NBR1 knockout,characterized by increased effector T lymphocytes and decreased exhausted T lym-phocytes(P＜0.01).Conclusion Using CRISPR/Cas9 technology,we construct a lung cancer mouse model with targeted NBR1 knockout.We verify that targeted inhibition of NBR1 expression significantly enhances the function-al activity of CD8+T lymphocytes in lung tissues,resulting in suppressed tumor growth,reduced tumor burden,and extended survival in lung cancer mice.This study lays an experimental foundation for investigations into the mechanisms and functions of NBR1 and other genes in lung adenocarcinoma cells.关键词
肺腺癌/AAV6/CRISPR/Cas9/NBR1/基因编辑/免疫治疗Key words
lung adenocarcinoma/AAV6/CRISPR/Cas9/NBR1/gene editing/immunotherapy分类
医药卫生引用本文复制引用
王博康,朱名扬,张秀森,孙江涛..基于AAV6-CRISPR-Cas9系统抑制NBR1表达对肺癌小鼠肿瘤免疫调控的影响[J].安徽医科大学学报,2024,59(12):2103-2111,9.基金项目
河南省重点研发与推广专项(科技攻关)项目(编号:242102310155) Key Research and Development Program of Henan Province(No.242102310155) （科技攻关）
