Construction of lentiviral vector for late endosomal/lysosomal adaptor, mitogen-activated protein kinase and mammalian target of rapamycin activator 2 interference and its role on inflammatory factor secretion of macrophages
10.3760/cma.j.issn.1000-6680.2019.10.005
- VernacularTitle: 晚期内含子/溶酶体适配子和促分裂原活化蛋白激酶以及哺乳动物重组雷帕霉素激活物分子2基因的RNA干扰慢病毒载体的构建及其对巨噬细胞中炎性因子分泌的影响
- Author:
Ting WU
1
;
Fangming XU
1
;
Cong SU
2
;
Yanyan LIU
3
;
Yanhu LAN
3
;
Jiabin LI
1
Author Information
1. Department of Infectious Diseases, the First Affiliated Hospital of Anhui Medical University, Hefei 230000, China
2. Department of Infectious Diseases, Chaohu Hospital of Anhui Medical University, Chaohu 238000, China
3. Anhui Center for Surveillance of Bacterial Resistance, Hefei 230000, China
- Publication Type:Journal Article
- Keywords:
Macrophages;
Klebsiella pneumoniae;
Gene silencing;
shRNA
- From:
Chinese Journal of Infectious Diseases
2019;37(10):605-609
- CountryChina
- Language:Chinese
-
Abstract:
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.
