本文選題：原代枯否細(xì)胞 + 干擾素調(diào)節(jié)因子　； 參考：《中國(guó)免疫學(xué)雜志》2016年04期

【摘要】：目的:探討干擾素調(diào)節(jié)因子3(Interferon regulator factor 3,IRF3)shRNA腺病毒對(duì)脂多糖(Lipopolysaccharide,LPS)刺激枯否細(xì)胞(Kupffer cell,KC)早期細(xì)胞因子分泌動(dòng)態(tài)變化的影響。方法:采用在體灌注分離培養(yǎng)大鼠原代KC,以IRF3shRNA腺病毒體外感染KC,48 h后采用LPS刺激細(xì)胞,于0、2、4和6 h收集細(xì)胞培養(yǎng)上清液,并收集6 h細(xì)胞。上清液細(xì)胞因子的分泌采用ELISA分析;細(xì)胞IRF3基因表達(dá)采用RT-PCR和Western blot方法檢測(cè)。結(jié)果:LPS刺激誘導(dǎo)了KC內(nèi)IRF3 mRNA和蛋白質(zhì)表達(dá)升高,IRF3 shRNA腺病毒的應(yīng)用抑制了LPS刺激誘導(dǎo)和非刺激組成性IRF3 mRNA和蛋白質(zhì)的表達(dá);KC受LPS刺激活化后極早期(2 h)IFN-β分泌即上升,4 h達(dá)峰值,6 h分泌水平開始下降,但仍維持于高水平。干擾腺病毒的應(yīng)用抑制了LPS刺激后各時(shí)間點(diǎn)IFN-β的分泌,抑制分泌高峰的出現(xiàn),并使6 h分泌水平趨于正常;KC活化后極早期即分泌大量TNF-α,并于2 h內(nèi)達(dá)到峰值,隨后分泌逐漸下降,但6 h仍維持于高水平。干擾腺病毒的應(yīng)用抑制了LPS刺激各時(shí)間點(diǎn)TNF-α的分泌,并抑制了分泌高峰的出現(xiàn);IL-1β分泌增高出現(xiàn)于LPS刺激4 h后,6 h分泌水平達(dá)更高值。干擾腺病毒的應(yīng)用抑制了LPS刺激早期KC對(duì)IL-1β的分泌;KC受LPS刺激活化后極早期IL-10分泌即上升,且隨著LPS刺激時(shí)間延長(zhǎng),其分泌水平逐漸增加。IRF3 shRNA腺病毒的應(yīng)用促進(jìn)了LPS刺激后早期各時(shí)間點(diǎn)IL-10的分泌。結(jié)論:IRF3 shRNA腺病毒可使原代枯否細(xì)胞IRF3基因表達(dá)沉默;在LPS刺激原代KC,IRF3可促進(jìn)其下游信號(hào)分子IFN-β、前炎細(xì)胞因子TNF-α和IL-1β的分泌,并抑制抑炎細(xì)胞因子IL-10分泌。因此,IRF3可能在肝組織免疫炎癥性損傷的發(fā)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- 偽 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- 偽 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- 偽 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.
【作者單位】： 南京醫(yī)科大學(xué)附屬上海松江中心醫(yī)院/上海交通大學(xué)附屬第一人民醫(yī)院松江分院感染科;
【基金】：國(guó)家自然科學(xué)基金項(xiàng)目(81070357,30660066) 上海市松江區(qū)科學(xué)技術(shù)攻關(guān)項(xiàng)目(14SJGGYY22)
【分類號(hào)】：R575.3

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