摘要
目的通过shRNA沉默肿瘤坏死因子受体相关因子6(tumour necrosis factor receptor associated factor 6,TRAF6)基因表达,研究TRAF6沉默基因对内毒素/D-氨基半乳糖(LPS/D-GalN)诱导的急性肝衰竭小鼠炎症反应的影响。方法通过体外实验筛选针对TRAF6基因的最有效小干扰RNA(siRNA)序列,采用流体力学注射法将pTRAF6-shRNA表达质粒转染BALB/c小鼠,24h后重复注射1次。第2次注射后24h,予LPS/D-GalN腹腔注射制备急性肝衰竭内毒素炎症模型。设正常对照组、模型对照组、空白质粒/LPS组及RNAi/LPS组。观察各组小鼠肝脏病理变化,Real-time PCR检测TRAF6、IL-6及COX-2mRNA的表达,ELISA检测血清TNF-α、IL-1β及TGF-β1水平变化,Western blot检测肝细胞TRAF6、NF-κB p65的表达。结果 Real-time PCR及Western blot检测显示,pTRAF6-shRNA1能有效沉默TRAF6mRNA及蛋白表达,其中TRAF6mRNA表达较正常小鼠下降约60.13%,TRAF6蛋白表达降低约52.08%,差异均有统计学意义(均P<0.01)。ELISA检测显示各组小鼠TNF-α、IL-1β均于造模后8h达到峰值,TGF-β1于16h达到峰值;RNAi/LPS组小鼠血清TNF-α、IL-1β及TGF-β1水平明显低于同时间点模型对照组。Real-time PCR结果显示RNAi/LPS组小鼠IL-6、COX-2及TRAF6mRNA表达下降,与模型对照组比较差异均有统计学意义(均P<0.01)。Western blot结果显示RNAi/LPS组小鼠肝组织总蛋白NF-κB p65表达水平显著高于正常对照组(P<0.05),与模型对照组无差异,而胞核NF-κB p65明显低于模型对照组(P<0.05)。结论 pTRAF6-shRNA可能通过下调NF-κB p65水平,抑制炎症相关细胞因子和炎性介质表达水平,从而减轻内毒素/半乳糖诱导的小鼠急性肝损伤。
Objective To examine the effect of silencing tumor necrosis factor（TNF）receptor-associated factor-6（TRAF6） on the inflammatory responses in mice with lipopolysaccharide （LPS）/D-galactosamine （GalN）-induced acute liver failure （ALF）.Methods The small interfering RNA（siRNA）sequence that could most effectively target TRAF6 was screened out in vitro. The plasmid pTRAF6-shRNA was delivered into BALB/c mice by hydrodynamics-based gene transfection（HGT）twice at an interval of 24 h.Twenty-four h after the second HGT,LPS/D-GalN was intraperitoneally administrated to BALB/c mice to establish the endotoxin-induced inflammatory model of acute liver failure.Animals were divided into four groups：normal con-trol group,model control group,blank plasmid/LPS group and RNAi/LPS group.Blood samples were collected at 4,8 and 16 h after LPS/D-GalN injection.HE staining was used to observe the pathological changes of liver tissues.Meanwhile,TRAF6,in-terleukin-6（IL-6）and cyclooxygenease（COX）-2 mRNA levels were detected by real-time PCR.Inflammatory cytokines[TNF-α, IL-1β,transforming growth factor （TGF）-β1 and IL-6 ]were measured by ELISA.Western blot analysis was used to detect TRAF6 and NF-κB p65 expression in the nuclear extracts of hepatocytes.Results Our results showed that TRAF6 mRNA and protein expressions were remarkably reduced by 60.13% and 52.08%,respectively,in the mouse liver（P〈0.01）.ELISA indi-cated that the secretions of TNF-α,IL-1βand TGF-β1 were markedly increased after LPS/D-GalN inj ection,with TNF-α,IL-1βpeaking at 8 h,and TGF-β1 at 1 6 h.Furthermore,the levels of TNF-α,IL-1βand TGF-β1 were lower in RNAi/LPS group than in model control group at different detection time points.Real-time PCR showed that IL-6 ,COX-2 and TRAF6 mRNA levels were obviously lower in RNAi/LPS group than in model control group（P〈0.01）.Moreover,total NF-κB p65 protein levels＆amp;nbsp;were effectively increased in RNAi/LPS group as compared with normal control group（P〈0.05
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2016年第3期278-283,310,共7页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
