晚期内体/溶酶体适配蛋白2(LAMTOR2)基因缺失加重肺炎克雷伯菌引起的小鼠脓毒症

LAMTOR2 deficiency exacerbates Klebsiella pneumoniae-induced liver sepsis in mice

目的建立肺炎克雷伯菌(K.pneumoniae)感染肝组织晚期内体/溶酶体适配蛋白2(LAMTOR2)基因条件敲除小鼠模型,初步探究LAMTOR2基因在K.pneumoniae感染引起肝脓毒症过程中的作用及机制。方法繁育获得肝脏LAMTOR2基因条件性敲除小鼠(LAMTOR2^(flox/flox);Alb⁃Cre^(+))和同窝阴性对照组小鼠(LAMTOR2^(flox/flox))。提取实验组和对照组小鼠的肝组织,利用实时荧光定量PCR和Western blot法检测LAMTOR2的mRNA和蛋白表达水平确定LAMTOR2在肝组织的敲除效果。实验组和对照组小鼠分别感染K.pneumoniae,观察24、36、48、72 h的小鼠生存率,HE染色观察肝组织病变情况;利用实时荧光定量PCR检测实验组和对照组小鼠感染K.pneumoniae 24 h后肝组织肿瘤坏死因子α(TNF⁃α)、白细胞介素1β(IL⁃1β)和CXC趋化因子配体1(CXCL1)的mRNA的表达水平。结果成功获得肝组织LAMTOR2基因条件性敲除小鼠;与对照组小鼠相比,实验组小鼠在感染K.pneumoniae后生存率降低,肝组织出现更严重损伤;实验组小鼠肝组织TNF⁃α、IL⁃1β和CXCL1的mRNA表达水平显著降低,小鼠免疫应答能力降低。结论LAMTOR2在K.pneumoniae感染诱导脓毒症的过程中起保护作用。

Objective To explore the biological function and mechanisms of LAMTOR2 during Klebsiella pneumoniae(K.pneumoniae)induced liver sepsis by establishing late endosomal/lysosomal adaptor 2(LAMTOR2)gene liver conditional knockout mouse model infected by K.pneumoniae.Methods LAMTOR2 gene liver conditional knockout mice(LAMTOR2^(flox/flox);Alb⁃Cre^(+))and littermate controls(LAMTOR2^(flox/flox))were generated and bred.LAMTOR2 gene knockout efficiency in liver was determined by real⁃time quantitative PCR(RT⁃qPCR)and Western blot analysis.Then,both group mice were infected with K.pneumoniae,and survival rates and liver pathological changes were determined.The expression levels of liver TNF⁃α,IL⁃1βand CXCL1 mRNA were detected by RT⁃qPCR.Results LAMTOR2 gene liver conditional knockout mice were generated and bred successfully;compared to the littermate controls,LAMTOR2^(flox/flox),Alb⁃Cre^(+) mice showed lower survival rates and more severe liver injury.The expression levels of TNF⁃α,IL⁃1βand CXCL1 mRNA were reduced in LAMTOR2^(flox/flox) and the ability of immune response was decreased in mice.Alb⁃Cre^(+) mice liver compared to these of littermate controls post K.pneumoniae infections.Conclusion LAMTOR2 plays a protective role during K.pneumoniae⁃induced liver sepsis.