Objective To construct lentiviral vector of late endosomal／lysosomal adaptor, mitogen-activated protein kinase and mammalian target of rapamycin activator 2 ( lamtor2) gene, and to explore its regulatory role on inflammatory response of macrophages after Klebsiella pneumoniae infection.Methods Two pairs of mouse lamtor2 short hairpin RNA (shRNA) were designed and sub-cloned into PLKO.1-puro to construct lentiviral vector, and were transfected into the murine RAW264.7 macrophage.There were two experimental groups including pLKO.1-puro shlamtor 2-1(sh1 group) and pLKO.1-puro shlamtor 2-2 (sh2 group), and the RAW264.7 cells transfected with non-treated pLKO.1-puro was set as control.The expession of lamtor2 were detected by real-time quantitative polymerase chain reaction ( RT-qPCR ) and Western blot.The levels of inflammatory factors including interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-αsecreted by the cells were detected by RT-qPCR.T test was used for comparison between groups.Results The recombinant lentiviral vector PLKO.1-shlamtor 2 transfected RAW264.7 cells successfully.The relative expressions of lamtor2 mRNA in the control group, the sh1 group and the sh2 group were 1.000 ±0.000, 0.596 ±0.125 and 0.120 ±0.080, respectively.The expression of lamtor2 in the sh2 group was significantly lower than that in the sh 1 group (t＝3.399, P＝0.015), and they were both significantly lower than the control group ( t ＝3.333 and 9.734, respectively, both P ＜0.05).After infection with Klebsiella pneumoniae, expression levels of IL-1β( t ＝15.20), IL-6 (t＝43.30) and TNF-α(t＝12.67) were significantly higher than those in the control group (all P＜0.01).Conclusion The lentiviral vector of lamtor2 can stably down-regulate the expression of lamtor2 gene in macrophages through RNA interference mechanism , which has a significant effect on the secretion of inflammatory factors of macrophages that are infected with Klebsiella pneumoniae.

Objective: To construct lentiviral vector of late endosomal/lysosomal adaptor, mitogen-activated protein kinase and mammalian target of rapamycin activator 2 (lamtor2) gene, and to explore its regulatory role on inflammatory response of macrophages after Klebsiella pneumoniae infection. Methods: Two pairs of mouse lamtor2 short hairpin RNA (shRNA) were designed and sub-cloned into PLKO.1-puro to construct lentiviral vector, and were transfected into the murine RAW264.7 macrophage. There were two experimental groups including pLKO.1-puro shlamtor 2-1(sh1 group) and pLKO.1-puro shlamtor 2-2 (sh2 group), and the RAW264.7 cells transfected with non-treated pLKO.1-puro was set as control. The expession of lamtor2 were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. The levels of inflammatory factors including interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α secreted by the cells were detected by RT-qPCR. T test was used for comparison between groups. Results: The recombinant lentiviral vector PLKO.1-shlamtor 2 transfected RAW264.7 cells successfully. The relative expressions of lamtor2 mRNA in the control group, the sh1 group and the sh2 group were 1.000±0.000, 0.596±0.125 and 0.120±0.080, respectively. The expression of lamtor2 in the sh2 group was significantly lower than that in the sh1 group (t=3.399, P=0.015), and they were both significantly lower than the control group (t=3.333 and 9.734, respectively, both P< 0.05). After infection with Klebsiella pneumoniae, expression levels of IL-1β (t=15.20), IL-6 (t=43.30) and TNF-α (t=12.67) were significantly higher than those in the control group (all P<0.01). Conclusion The lentiviral vector of lamtor2 can stably down-regulate the expression of lamtor2 gene in macrophages through RNA interference mechanism, which has a significant effect on the secretion of inflammatory factors of macrophages that are infected with Klebsiella pneumoniae.

Objective : To establish the liver conditional knockout mouse model ofNod2gene infected byKlebsiella pneumoniae(K.pneumoniae),and to explore the role and mechanism ofNod2gene in the process of liver abscess caused byK.pneumoniaeinfections. Methods : Nod2flox/floxmice were obtained by self-crossing ofNod2flox/+mice, andAlb-Cre+mice were hybridized withNod2flox/+to obtainNod2flox/+;Alb-Cre+mice, then the above two genotypes mice were crossed to obtain liver conditional knockout mice ofNod2gene(Nod2flox/flox;Alb-Cre+) and negative control mice in the same litter(Nod2flox/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 ofNod2gene in the liver.Both experimental group and control group mice were infected withK.pneumoniae,and the survival rate and pathological changes of livers were observed at different time points, and mRNA expression levels of Tumor necrosis factor α(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 24 h postK.pneumoniaeinfections. Results : The expression of NOD2 mRNA in the liver ofNod2flox/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 withK.pneumoniaein the experimental group decreased(median survival time=60.5 h,P=0.046 9) and the liver tissue showed more serious pathological damage, furthermore the mRNA expression levels of TNF-α,IL-1β and 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 byK.pneumoniaeinfections.