【摘要】：文章旨在探索核因子-κB(NF-κB)和絲裂原活化蛋白激酶(MAPKs)通路是否介導(dǎo)益生性釀酒酵母菌(Saccharomyces cerevisiae)誘導(dǎo)綿羊瘤胃上皮細(xì)胞(RECs)β-防御素-1(SBD-1)基因的轉(zhuǎn)錄。首先建立綿羊RECs培養(yǎng)體系作為體外試驗?zāi)Ｐ?選用誘導(dǎo)SBD-1轉(zhuǎn)錄最高的菌液濃度和誘導(dǎo)培養(yǎng)時間進(jìn)行信號通路初步研究,采用實時熒光定量逆轉(zhuǎn)錄PCR(RT-qPCR)對已建立的誘導(dǎo)SBD-1轉(zhuǎn)錄模型中的細(xì)胞膜受體——Toll樣受體2(TLR2)、信號銜接蛋白——髓樣分化因子(MyD88)以及NF-κB和MAPKs通路中的相關(guān)因子基因轉(zhuǎn)錄變化進(jìn)行檢測;然后選用NF-κB和MAPKs通路中的4種特異性抑制劑(即NF-κB通路特異性抑制劑PDTC、P38通路特異性抑制劑SB202190、ERK 1/2通路特異性抑制劑PD98059、JNK通路特異性抑制劑SP600125)通過單獨(dú)或相互組合處理細(xì)胞后再進(jìn)行誘導(dǎo)培養(yǎng),同時采用RT-qPCR的方法檢測用抑制劑處理綿羊RECs后SBD-1mRNA的轉(zhuǎn)錄水平。結(jié)果表明:釀酒酵母菌刺激RECs后,NF-κB和MAPKs通路中各因子NF-κB、P38、JNK、ERK1/2、細(xì)胞膜受體TLR2與信號銜接蛋白MyD88的mRNA水平與未刺激組相比均有所升高,且呈顯著性差異(P0.01或P0.05);通過單獨(dú)或組合添加抑制劑后再誘導(dǎo),均發(fā)現(xiàn)特異性抑制劑PDTC、SB202190、SP600125、PD98059可極顯著抑制釀酒酵母菌對RECs SBD-1的上調(diào)作用(P0.01),且P38通路特異性抑制劑SB202190的抑制效果最明顯。結(jié)果提示,釀酒酵母菌誘導(dǎo)綿羊RECs SBD-1的轉(zhuǎn)錄可能與TLR2-MyD88-NF-κB/MAPKs通路有關(guān),但以TLR2-MyD88-MAPKs中的TLR2-MyD88-P38通路為主要的信號通路。
[Abstract]:The aim of this study was to investigate whether nuclear factor- 魏 B (NF- 魏 B) and mitogen-activated protein kinase (MAPKs) pathway mediate the transcription of (RECs) 尾 -defensin-1 (SBD-1) gene in sheep rumen epithelial cells induced by (Saccharomyces cerevisiae). Firstly, sheep RECs culture system was established as an in vitro experimental model. The highest concentration of bacterial fluid and the time of inducing SBD-1 transcription were selected to study the signal pathway. The transcriptional changes of cell membrane receptor Toll like receptor 2 (TLR2), signal junction protein-myeloid differentiation factor (MyD88), NF- 魏 B and MAPKs pathway were detected by real-time fluorescence quantitative reverse transcription PCR (RT-qPCR). Then the four specific inhibitors of NF- 魏 B and MAPKs pathway (that is, NF- 魏 B pathway specific inhibitor, PDTC,P38 pathway specific inhibitor, SB202190,ERK 1 / 2 pathway specific inhibitor, PD98059,JNK pathway specific inhibitor SP600125) were treated separately or in combination with each other. Cells were then induced and cultured, At the same time, RT-qPCR was used to detect the transcription level of SBD-1mRNA in sheep treated with RECs. The results showed that after RECs was stimulated by Saccharomyces cerevisiae, the mRNA levels of NF- 魏 B and NF- 魏 B P38, JNKK ERK 1 / 2 in the NF- 魏 B and MAPKs pathway were increased, and the mRNA levels of the membrane receptor TLR2 and the signal junction protein MyD88 were higher than those of the unstimulated group. The specific inhibitor PDTC,SB202190,SP600125,PD98059 could significantly inhibit the up-regulation of RECs SBD-1 by Saccharomyces cerevisiae (P0.01), and the inhibition effect of P38 pathway specific inhibitor SB202190 was the most obvious. The results suggest that the transcription of RECs SBD-1 in sheep induced by Saccharomyces cerevisiae may be related to the TLR2-MyD88-NF- 魏 B/MAPKs pathway, but the TLR2-MyD88-P38 pathway in TLR2-MyD88-MAPKs is the main signal pathway.
【作者單位】： 內(nèi)蒙古農(nóng)業(yè)大學(xué)獸醫(yī)學(xué)院;農(nóng)業(yè)部動物疾病臨床診療技術(shù)重點(diǎn)實驗室;
【基金】：國家自然科學(xué)基金(31160491;31560682)
【分類號】：S826

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