IRF3基因干扰对LPS刺激原代枯否细胞早期细胞因子分泌动态变化的影响  

Effects of IRF3 gene interference on dynamic changes of cytokine secretions in early of LPS stimulation in primary Kupffer cells

作　　者：朱彤[1] 涂文娟[1] 谈志丽 刘亮明[1] 

机构地区：[1]南京医科大学附属上海松江中心医院/上海交通大学附属第一人民医院松江分院感染科,上海201600

出　　处：《中国免疫学杂志》2016年第4期470-475,479,共7页Chinese Journal of Immunology

基　　金：国家自然科学基金项目(81070357;30660066);上海市松江区科学技术攻关项目(14SJGGYY22)

摘　　要：目的:探讨干扰素调节因子3(Interferon regulator factor 3,IRF3)shRNA腺病毒对脂多糖(Lipopolysaccharide,LPS)刺激枯否细胞(Kupffer cell,KC)早期细胞因子分泌动态变化的影响。方法:采用在体灌注分离培养大鼠原代KC,以IRF3shRNA腺病毒体外感染KC,48 h后采用LPS刺激细胞,于0、2、4和6 h收集细胞培养上清液,并收集6 h细胞。上清液细胞因子的分泌采用ELISA分析;细胞IRF3基因表达采用RT-PCR和Western blot方法检测。结果:LPS刺激诱导了KC内IRF3 mRNA和蛋白质表达升高,IRF3 shRNA腺病毒的应用抑制了LPS刺激诱导和非刺激组成性IRF3 mRNA和蛋白质的表达;KC受LPS刺激活化后极早期(2 h)IFN-β分泌即上升,4 h达峰值,6 h分泌水平开始下降,但仍维持于高水平。干扰腺病毒的应用抑制了LPS刺激后各时间点IFN-β的分泌,抑制分泌高峰的出现,并使6 h分泌水平趋于正常;KC活化后极早期即分泌大量TNF-α,并于2 h内达到峰值,随后分泌逐渐下降,但6 h仍维持于高水平。干扰腺病毒的应用抑制了LPS刺激各时间点TNF-α的分泌,并抑制了分泌高峰的出现;IL-1β分泌增高出现于LPS刺激4 h后,6 h分泌水平达更高值。干扰腺病毒的应用抑制了LPS刺激早期KC对IL-1β的分泌;KC受LPS刺激活化后极早期IL-10分泌即上升,且随着LPS刺激时间延长,其分泌水平逐渐增加。IRF3 shRNA腺病毒的应用促进了LPS刺激后早期各时间点IL-10的分泌。结论:IRF3 shRNA腺病毒可使原代枯否细胞IRF3基因表达沉默;在LPS刺激原代KC,IRF3可促进其下游信号分子IFN-β、前炎细胞因子TNF-α和IL-1β的分泌,并抑制抑炎细胞因子IL-10分泌。因此,IRF3可能在肝组织免疫炎症性损伤的发生中起中心作用。Objective： To investigate the effects of IRF3 shRNA adenovirus on dynamic changes of early cytokines in LPS-stimulated primary Kupffer cells（ KCs）. Methods： Rat KCs were isolated and purified by means of in situ perfusion. After being infected with adenovirus carrying IRF3 shRNA for 48 h,KCs were stimulated with LPS. Cell culture supernatants were collected respectively at0,2,4 and 6 h after LPS stimulation as well as cells at 6 h. Supernatant cytokine secretion levels were detected by enzyme-linked immunosorbent assay（ ELISA）. Intracellular gene expressions were tested by RT-PCR and Westeron blot. Results： IRF3 mRNA and protein were induced by LPS,but suppressed by IRF3 shRNA adenovirus in LPS-stimulated or non-stimulated KCs. IFN-β secretions rose in the very early stage（ at 2 h）,reached the peak at 4 h,and began to reduce but still remained high levels at 6 h after LPS stimulation in KCs. Interference adenovirus pretreatment suppressed IFN-β secretions（ especially the secretion peak） at each time point after LPS stimulation. IFN-β secretions reached normal levels at 6 h after the stimulation in adenovirus-pretreated cells; TNF-α secretions rapidly increased in the very early stage and reached the peak at 2 h,then began to decrease gradually,but remained high levels at 6 h after LPS stimulation in KCs. Interference adenovirus pretreatment inhibited LPS-induced TNF-α secretions,especially the secretion peak; IL-1βsecretions did not increase untill 4 h,but reached a higher level at 6 h after LPS stimulation. Interference adenovirus suppressed IL-1βsecretions in the early stage of LPS stimulation; IL-10 secretions began to rise in the very early stage,and gradually increased over time after LPS stimulation in KCs. Pretreatment of adenovirus with IRF3 shRNA promoted upregulations of IL-10 secretions at each time point of the early of LPS stimulation. Conclusion： IRF3 gene expression can be silenced by IRF3 shRNA adenovirus. IRF3 can promote its downstream signaling molecule IFN-β and

关 键 词：原代枯否细胞 干扰素调节因子3 基因干扰 固有免疫 炎性反应 

分 类 号：R575.3[医药卫生—消化系统]