Cc10基因敲除小鼠模型的构建及气道炎症表型分析  

Construction of a Cc10 Gene Knockout Mouse Model and Phenotypic Analysis of Airway Inflammation

作　　者：龙洁 魏丹丹 吴全龙 赵子莹 张亚亚 陈艳焦 杨永清 徐玉东[1,2] LONG Jie;WEI Dandan;WU Quanlong;ZHAO Ziying;ZHANG Yaya;CHEN Yanjiao;YANG Yongqing;XU Yudong(Yueyang Hospital of Integrated Traditional Chinese and Western Medicine Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200437,China;Shanghai Research Institute of Acupuncture and Meridian,Shanghai 200030,China)

机构地区：[1]上海中医药大学附属岳阳中西医结合医院,上海200437 [2]上海市针灸经络研究所,上海200030

出　　处：《中国细胞生物学学报》2025年第2期240-249,共10页Chinese Journal of Cell Biology

基　　金：国家自然科学基金(批准号:81973952、82105013、82374583);上海市自然科学基金(批准号:23ZR1459900)资助的课题。

摘　　要：该研究旨在构建Cc10基因敲除小鼠模型,并利用屋尘螨(HDM)诱导的哮喘模型,分析Cc10基因缺失小鼠的气道炎症表型。采用CRISPR/Cas9技术,设计并体外转录针对Cc10基因2号外显子的sgRNA和Cas9表达载体,显微注射至受精卵后获得F0代基因敲除小鼠,通过进一步繁育和筛选,最终获得稳定遗传的Cc10^(-/-)小鼠。利用HDM致敏和激发诱导小鼠过敏性哮喘模型,通过分析肺泡灌洗液(BALF)中白细胞及其分类计数、2型炎性相关细胞因子、肺组织PAS染色及Muc5ac mRNA的表达水平来评估Cc10基因缺失对小鼠气道炎症表型的影响。PCR扩增及测序结果显示,Cc10基因被成功敲除。Western blot分析表明,Cc10^(-/-)小鼠肺组织中CC10蛋白表达缺失。Cc10^(-/-)哮喘模型小鼠BALF中白细胞总数显著高于野生型(WT)哮喘模型组,且各分类细胞(中性粒细胞、淋巴细胞、嗜酸性粒细胞、巨噬细胞和嗜碱性粒细胞)数量均显著增加。Cc10^(-/-)哮喘模型小鼠BALF中2型炎性相关细胞因子IL-4、IL-5、IL-13水平显著高于野生型(WT)哮喘模型组。肺组织PAS染色结果显示,Cc10^(-/-)哮喘模型小鼠具有更显著的支气管壁增厚、杯状细胞增生等病理变化,PAS染色评分显著高于WT哮喘模型组。PCR结果显示,Cc10^(-/-)哮喘模型小鼠肺组织Muc5ac mRNA表达水平显著高于WT哮喘模型组。该研究成功构建了Cc10基因敲除小鼠模型,并证明了Cc10基因缺失导致哮喘小鼠气道炎症加重,气道黏液分泌增多,提示CC10在调控气道炎症中发挥重要作用。该模型将为探索CC10在哮喘及其他呼吸系统疾病中的作用机制提供重要工具。This study aimed to construct a Cc10 gene knockout mouse model and analyzed the airway inflammatory phenotype of Cc10-deficient mice using a HDM(house dust mite)-induced asthma model.CRISPR/Cas9technology was employed to design and transcribe sgRNA(single guide RNA)targeting exon 2 of the Cc10 gene,along with Cas9 expression vectors in vitro.Fertilized eggs were microinjected with these components,resulting in the generation of F0 knockout mice.The allergic asthma model was then developed in these mice by sensitization and challenge with HDM.The impact of Cc10 gene deficiency on airway inflammatory phenotypes was evaluated by assessing leukocyte counts and their classified counts,type 2 inflammation-associated cytokines in BALF(bronchoalveolar lavage fluid),performing PAS staining on lung tissues,and measuring Muc5ac mRNA expression levels.PCR amplification and sequencing confirmed the successful knockout of the Cc10 gene.Western blot analysis demonstrated the absence of CC10 protein expression in the lung tissues of Cc10^(–/–)mice.The total leukocyte count in BALF of Cc10^(–/–)asthmatic mice was significantly higher than that of the WT(wild-type)counterparts,with notable increases observed in neutrophils,lymphocytes,eosinophils,macrophages,and basophils.The levels of type 2 inflammation-associated cytokines IL-4,IL-5,and IL-13 were significantly higher in the BALF of Cc10^(–/–)asthma model mice than in the WT asthma model group.PAS staining of lung tissues revealed that the Cc10^(–/–)asthmatic mice exhibited significant pathological changes,including thickened bronchial walls and goblet cell hyperplasia,with PAS staining scores markedly higher than those in WT asthmatic mice.PCR analysis showed that Muc5ac m RNA expression levels in the lung tissues of Cc10^(–/–)asthmatic mice were significantly elevated compared to WT counterparts.Collectively,this study successfully constructed a Cc10 gene knockout mouse model and demonstrated that Cc10 deficiency exacerbates airway inflammation and enhance

关 键 词：CC10 CRISPR/Cas9 基因敲除小鼠 哮喘 炎症 MUC5AC 

分 类 号：R562.25[医药卫生—呼吸系统] R-332[医药卫生—内科学]