摘要
目的构建针对晚期内含子/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(late endosomal/lysosomal adaptor,mitogen-activated protein kinase and mammalian target of rapamycin activator 2,lamtor2)基因的小发夹RNA慢病毒载体,探讨RNA干扰lamtor2基因表达后对肺炎克雷伯菌感染引起的巨噬细胞中炎性因子分泌的调控作用.方法针对小鼠lamtor2基因设计2对小发夹RNA(shlamtor2),重组构建慢病毒载体pLKO.1-puro shlamtor2,并感染小鼠巨噬细胞系RAW264.7细胞.设2个实验组,pLKO.1-puro shlamtor2-1感染组(sh1组)和pLKO.1-puro shlamtor2-2感染组(sh2组),以空载pLKO.1质粒感染RAW264.7细胞为对照组.通过实时定量PCR和蛋白质印迹法检测各组细胞中lamtor2基因表达.肺炎克雷伯菌感染sh2组细胞,采用实时定量PCR方法检测细菌感染后细胞内白细胞介素(interleukin,IL)-1β、IL-6和肿瘤坏死因子(tumor necrosis factor,TNF)-α表达水平的变化.2组间比较采用t检验.结果重组慢病毒颗粒pLKO.1-shlamtor2感染RAW 264.7细胞后,对照组lamtor2基因mRNA相对表达量为1.000±0.000,sh1组为0.596±0.125,sh2组为0.120±0.080,sh2组的lamtor2表达量低于sh1组,差异有统计学意义(t=3.399,P=0.015),且sh1组和sh2组的表达量均低于对照组,差异均有统计学意义(t=3.333、9.734,均P<0.05).以肺炎克雷伯菌感染的野生RAW 264.7细胞作为对照,sh2组细胞感染肺炎克雷伯菌后,细胞中的IL-1β(t=15.20)、IL-6(t=43.30)和TNF-α(t=12.67)表达水平均高于对照组(均P<0.01).结论针对lamtor2基因的小发夹RNA可通过RNA干扰机制稳定下调巨噬细胞中lamtor2基因的表达,基因下调对感染了肺炎克雷伯菌的巨噬细胞内的炎性因子的分泌有明显的促进作用.
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.
作者
伍婷
徐方明
苏丛
刘艳艳
兰燕虎
李家斌
Wu Ting;Xu Fangming;Su Cong;Liu Yanyan;Lan Yanhu;Li Jiabin(Department of Infectious Diseases,the First Affiliated Hospital of Anhui Medical University,Hefei 230000,China;Anhui Center for Surveillance of Bacterial Resistance,Hefei 230000,China;Department of Infectious Diseases,Chaohu Hospital of Anhui Medical University,Chaohu 238000,China)
出处
《中华传染病杂志》
CAS
CSCD
2019年第10期605-609,共5页
Chinese Journal of Infectious Diseases
基金
国家自然科学基金(81673242)。
