巨噬细胞Lamtor2基因敲除小鼠对肺炎克雷伯菌易感性的研究  

Study on susceptibility of macrophage Lamtor2 knockout mice to Klebsiella pneumoniae

作　　者：苏丛 伍婷 徐方明 陈昊然 刘艳艳[2,3,4] 律娜 兰燕虎[3,4] 李家斌 Su Cong;Wu Ting;Xu Fangming(Dept of Infectious Diseases,The Chaohu Affiliated Hospital of Anhui Medical University,Chaohu 238000;Dept of Infectious Diseases,The First Affiliated Hospital of Anhui Medical University,Hefei 230022)

机构地区：[1]安徽医科大学附属巢湖医院感染病科,巢湖238000 [2]安徽医科大学第一附属医院感染病科,合肥230022 [3]安徽省细菌耐药性监控中心,合肥230022 [4]安徽医科大学细菌耐药研究所,合肥230022

出　　处：《安徽医科大学学报》2021年第4期505-509,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81673242、81973983);2018年度安徽高校自然科学研究项目(编号:KJ2018A0193);2018安徽省高校优秀拔尖人才培育(编号:gxyq2018010)。

摘　　要：目的通过建立Lamtor2巨噬细胞条件敲除小鼠感染肺炎克雷伯菌(Kp)模型,初步研究Lamtor2基因生物学功能及其在Kp感染过程中的相关机制。方法将引进的Lamtor2^(flox/+)小鼠自交,Lyz2-Cre小鼠与Lamtor2^(flox/+)进行杂交,获得基因型为Lamtor2^(flox/flox)Lyz2-Cre^(-/-)和Lamtor2^(flox/+)Lyz2-Cre^(+/-)的子代小鼠;将上述两种基因型子代小鼠进行杂交,得到对照组小鼠(Lamtor2^(flox/flox)Lyz2-Cre^(-/-))和Lamtor2条件性基因敲除小鼠(Lamtor2^(flox/floxLyz)2-Cre^(+/-))。提取小鼠脚趾基因组DNA,经聚合酶链式反应(PCR)扩增,通过琼脂糖凝胶电泳鉴定子代小鼠基因型并提取肺泡巨噬细胞,利用qRT-PCR和Western blot验证Lamtor2基因敲除效果。小鼠感染Kp,观察生存状况;体外采用RT-qPCR检测Kp感染肺泡巨噬细胞肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β和Cxcl1表达水平,两组间比较采用t检验;Western blot验证iNOS的表达水平。结果成功建立Lamtor2条件性基因敲除小鼠模型,子代存活且可育;与对照组小鼠比较,实验组小鼠在感染Kp后生存率降低;Lamtor2^(flox/flox)Lyz2-Cre^(+/-)小鼠来源的肺泡巨噬细胞中的TNF-α(t=17.54,P=0.0032)、IL-1β(t=15.37,P=0.0042)和Cxcl1(t=37.82,P=0.0007)表达水平降低,且iNOS表达下降。结论构建了Lamtor2条件性基因敲除小鼠,感染肺炎克雷伯菌后,小鼠的生存率下降;肺泡巨噬细胞iNOS表达降低。Objective To study the biological function of Lamtor2 gene and its mechanism in the process of Lamtor2 infection,the model of Lamtor2 macrophage conditioned knockout mice infected with KP was established.Methods Lamtor2^(flox/+)mice were introduced for self-crossing,and Lyz2-Cre mice were hybridized with Lamtor2^(flox/+)to obtain offspring mice with Lamtor2^(flox/flox)Lyz2-Cre^(-/-)and Lamtor2^(flox/+)Lyz2-Cre^(+/-).The two genotypes were hybridized to obtain control mice(Lamtor2^(flox/flox)Lyz2-Cre^(-/-))and lamtor2 conditional knockout mice(Lamtor2^(flox/flox)Lyz2-Cre^(+/-)).The toe genomic DNA of mice was extracted and amplified by polymerase chain reaction(PCR).The genotypes of offspring mice were identified by agar-gel electrophoresis and alveolar macrophages were extracted.The Lamtor2 gene knockout effect was verified by RT-qPCR and Western blot.Mice were infected with Kp and their survival conditions were observed.The expression levels of TNF-α,IL-1βand Cxcl1 after Kp infection with alveolar macrophages were detected by qRT-PCR in vitro,and the comparison between the two groups was performed by t test.Western blot was used to verify the expression level of iNOS.Results Lamtor2 conditional gene knockout mouse model was successfully established with viable and fertile offspring.Compared with control mice,experimental mice had lower survival rate after Kp infection.The levels of TNF-α(t=17.54,0.44±0.050,P=0.0032),IL-1β(t=15.37,0.35±0.043,P=0.0042),and Cxcl1(t=37.82,0.23±0.043,P=0.0007)from Lamtor2^(flox/flox) Lyz2-Cre^(+/-)mouse alveolar macrophages reduced,and the iNOS expression decreased.Conclusion Lamtor2 knockout mice are constructed,and the survival rate of mice and iNOS expression of alveolar macrophges decreases after infected with Kp.

关 键 词：Lamtor2 条件性基因敲除 繁育 肺炎克雷伯菌 

分 类 号：R332[医药卫生—人体生理学]