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

发布时间：2018-05-27 18:05

本文选题：原代枯否细胞 + 干扰素调节因子　；参考：《中国免疫学杂志》2016年04期

【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。
[Abstract]:Aim: to investigate the effect of interferon regulatory factor 3(Interferon regulator factor 3 (IRF3) shRNA adenovirus on the dynamic changes of cytokine secretion in Kupffer cell (KC) stimulated by lipopolysaccharide (LPS). Methods: primary rat KC cells were isolated and cultured by perfusion in vivo. The cells were stimulated by LPS for 48 h after infection with IRF3shRNA adenovirus in vitro. The supernatants were collected at 4 and 6 hours after infection with IRF3shRNA adenovirus, and the cells were collected for 6 h. The secretion of cytokines in supernatant was analyzed by ELISA and the expression of IRF3 gene was detected by RT-PCR and Western blot. Results the expression of IRF3 mRNA and protein in KC was induced by 1: LPS-stimulated. The application of IRF3 shRNA adenovirus inhibited the expression of IRF3 mRNA and protein in KC induced by LPS and non-stimulative components. The secretion of IFN- 尾 increased at the very early stage after the activation of KC stimulated by LPS. The level of secretion began to decrease at 4 h and 6 h. But it remains at a high level. The application of interfering adenovirus inhibited the secretion of IFN- 尾 at different time points after stimulation of LPS, inhibited the peak of secretion, and made the secretion level of 6 h tend to secrete a large amount of TNF- 伪 in the early stage after normal activation of KC, and reached the peak value within 2 h. Then the secretion decreased gradually, but remained at a high level at 6 h. The application of interfering adenovirus inhibited the secretion of TNF- 伪 at different time points stimulated by LPS, and inhibited the peak of secretion. The increase of IL-1 尾 secretion occurred at 6 h after LPS stimulation. The application of interfering adenovirus inhibited the secretion of IL-1 尾 by KC in the early stage of LPS stimulation. After activation by LPS, the secretion of IL-10 increased in the very early stage, and with the prolongation of the time of LPS stimulation, the secretion of KC increased in the early stage. Its secretion level increased gradually. IRF3 shRNA adenovirus promoted the secretion of IL-10 at different time points after LPS stimulation. Conclusion the expression of IRF3 gene in Kupffer cells was silenced by the adenovirus of 1: IRF3 shRNA, which stimulated the secretion of IFN- 尾, proinflammatory cytokines TNF- 伪 and IL-1 尾, and inhibited the secretion of anti-inflammatory cytokine IL-10 in primary Kupffer cells by LPS stimulation. Therefore, IRF3 may play a central role in the development of immune inflammatory injury in liver tissue.
【作者单位】：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;
【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)
【分类号】：R575.3

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