Nod2基因在肺炎克雷伯菌肝脓肿中的作用研究

Study of the role of Nod2 gene in Klebsiella pneumoniae liver abscess

目的建立肺炎克雷伯菌(K.pneumoniae)感染Nod2基因肝组织条件敲除小鼠模型,初步探究Nod2基因在K.pneumoniae感染引起肝脓肿过程中的作用及机制。方法将引进的Nod2^(flox/+)小鼠自交获得Nod2^(flox/flox)小鼠,同时将Alb-Cre^(+)小鼠与Nod2^(flox/+)进行杂交获得Nod2^(flox/+);Alb-Cre^(+)的子代小鼠;将上述两种基因型的子代小鼠进行杂交,获得Nod2基因肝脏条件性敲除小鼠(Nod2^(flox/flox);Alb-Cre^(+))和同窝阴性对照组小鼠(Nod2^(flox/flox))。提取小鼠脚趾基因组DNA,经PCR扩增,通过琼脂糖凝胶电泳鉴定子代小鼠基因型并提取小鼠肝脏,利用RT-qPCR和Western blot验证Nod2基因在肝脏中的敲除效果。实验组和对照组小鼠分别感染K.pneumoniae,观察不同时间点小鼠生存率及肝组织病理变化;利用RT-qPCR检测实验组和对照组小鼠感染K.pneumoniae 48 h后肝组织内肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)和趋化因子CXC配体1(CXCL1)的mRNA的表达水平。结果Nod2^(flox/flox);Alb-Cre^(+)小鼠肝脏NOD2 mRNA表达量降低(P<0.05),Western blot结果显示NOD2蛋白表达量降低;与对照组小鼠比较,实验组小鼠在感染K.pneumoniae后生存率降低(中位生存时间60.5 h,P=0.0469),肝组织出现了更为严重的病理损伤;实验组小鼠肝组织中TNF-α、IL-1β和CXCL1的mRNA表达水平降低,差异有统计学意义(P<0.05)。结论NOD2在K.pneumoniae感染诱导肝脓肿的过程中起保护作用。

Objective To establish the liver conditional knockout mouse model of Nod2 gene infected by Klebsiella pneumoniae(K.pneumoniae),and to explore the role and mechanism of Nod2 gene in the process of liver abscess caused by K.pneumoniae infections.Methods Nod2^(flox/flox) mice were obtained by self-crossing of Nod2^(flox/+)mice,and Alb-Cre^(+)mice were hybridized with Nod2^(flox/+)to obtain Nod2^(flox/+);Alb-Cre^(+)mice,then the above two genotypes mice were crossed to obtain liver conditional knockout mice of Nod2 gene(Nod2^(flox/flox);Alb-Cre^(+))and negative control mice in the same litter(Nod2^(flox/flox)).The genomic DNA of mice toe was extracted and amplified by polymerase chain reaction(PCR).The genotypes of offspring were identified by agar-gel electrophoresis and the livers of mice were extracted.Real-time fluorescence quantitative PCR(RT-qPCR)and Western blot were used to verify the knockout efficiency of Nod2 gene in the liver.Both experimental group and control group mice were infected with K.pneumoniae,and the survival rate and pathological changes of livers were observed at different time points,and mRNA expression levels of Tumor necrosis factorα(TNF-α),interleukin 1β(IL-1β)and C-X-C motif chemokine ligand 1(CXCL1)in the livers of experimental group and control group were detected by RT-qPCR 24h post K.pneumoniae infections.Results The expression of NOD2 mRNA in the liver of Nod2^(flox/flox);Alb-Cre^(+)mice decreased,and the Western blot results showed that the expression of NOD2 protein decreased.Compared with the control group,the survival rate of mice infected with K.pneumoniae in the experimental group decreased(median survival time=60.5 h,P=0.0469)and the liver tissue showed more serious pathological damage,furthermore the mRNA expression levels of TNF-α,IL-1βand CXCL1 in the livers of experimental group were lower than those of the control group,and the difference was statistically significant(P<0.05).Conclusion NOD2 plays a protective role in the process of liver abscess induced by K.pneumoniae infections.