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

作　　者：王博康 朱名扬 张秀森 孙江涛 Wang Bokang;Zhu Mingyang;Zhang Xiusen;Sun Jiangtao(Clinical Medical College of Henan University of Science and Technology,Luoyang 471000;Henan Key Laboratory of Tumor Epigenetics,Luoyang 471000;Cancer Institute of Henan University of Science and Technology,Luoyang 471000;Dept of Thoracic Oncology Surgery,Cancer Hospital of The First Affiliated Hospital of Henan University of Science and Technology,Luoyang 471000)

机构地区：[1]河南科技大学临床医学院,洛阳471000 [2]河南省肿瘤表观遗传重点实验室,洛阳471000 [3]河南科技大学肿瘤研究所,洛阳471000 [4]河南科技大学第一附属医院肿瘤医院胸部肿瘤外科,洛阳471000

出　　处：《安徽医科大学学报》2024年第12期2103-2111,共9页Acta Universitatis Medicinalis Anhui

基　　金：河南省重点研发与推广专项(科技攻关)项目(编号:242102310155)。

摘　　要：目的以腺相关病毒(AAV)为载体,利用CRISPR-Cas9系统靶向抑制BRCA1邻近基因1(NBR1)表达,建立NBR1基因敲除的肺癌小鼠模型,并探究其对肿瘤生长和免疫细胞浸润及功能活性的影响。方法利用在线网站CRISPOR(http://crispor.tefor.net/crispor.py)进行靶向鼠源NBR1基因(Gene ID:17966)的sgRNAs设计。使用AAV6作为sgRNAs的载体,使用PCR和DNA测序的方法确认重组病毒载体是否构建成功及确定基因敲除效率。为了确定小鼠体内最佳的AAV侵染方式,将6只C57BL/6J小鼠随机分为滴鼻组和气管内注射组,侵染28天后,通过观察小鼠肺组织冰冻切片的增强型绿色荧光蛋白表达情况,选择更有效的侵染方式进行后续实验。通过HE染色、免疫组化、组织免疫荧光和流式细胞术等方法检测小鼠肺部肿瘤生长情况以及肿瘤组织免疫细胞浸润和激活状态。结果DNA测序及荧光显微镜观察显示,AAV6-U6-sgNBR1-CAG-Cre-GFP病毒载体构建成功,且敲除效率稳定。荧光显微镜观察显示气管内给药法小鼠肺部感染病毒的效率较高(P<0.05)。HE染色结果显示靶向敲除NBR1后小鼠肺部肿瘤面积较对照组减少(P<0.01)。组织免疫荧光及流式细胞术结果显示,靶向敲除NBR1的肺癌小鼠肺癌组织中CD8^(+)T淋巴细胞功能活性较对照组增强,表现为效应型T淋巴细胞增多、耗竭型T淋巴细胞减少(P<0.01)。结论通过CRISPR/Cas9技术成功构建了靶向NBR1基因敲除的肺癌小鼠模型,验证了靶向抑制NBR1表达可明显增强肺组织CD8^(+)T淋巴细胞功能活性,使肿瘤生长受到抑制,减轻肿瘤负荷,延长肺癌小鼠生存期。为后续研究NBR1及其他基因在肺腺癌细胞中的作用机制和功能奠定了实验基础。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 experiments.Lung tumor growth,as well as immune cell infiltration and activation status in tumor tissues,were detected using methods including HE staining,immunohistochemistry,immunofluorescence,and flow cytometry.Results DNA sequencing and immunofluorescence results indicated successful construction of the AAV6-U6-sgNBR1-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 lymphocytes(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 functional activity of CD8^(+)T lymphocytes in lung tissues,resulting in suppressed tumor growth,reduced tumor burden,and exte

关 键 词：肺腺癌 AAV6 CRISPR/Cas9 NBR1 基因编辑 免疫治疗 

分 类 号：R734.2[医药卫生—肿瘤]