酿酒酵母菌诱导绵羊瘤胃上皮细胞SBD-1表达的信号通路初步研究

发布时间：2018-10-22 14:27

【摘要】：文章旨在探索核因子-κB(NF-κB)和丝裂原活化蛋白激酶(MAPKs)通路是否介导益生性酿酒酵母菌(Saccharomyces cerevisiae)诱导绵羊瘤胃上皮细胞(RECs)β-防御素-1(SBD-1)基因的转录。首先建立绵羊RECs培养体系作为体外试验模型,选用诱导SBD-1转录最高的菌液浓度和诱导培养时间进行信号通路初步研究,采用实时荧光定量逆转录PCR(RT-qPCR)对已建立的诱导SBD-1转录模型中的细胞膜受体——Toll样受体2(TLR2)、信号衔接蛋白——髓样分化因子(MyD88)以及NF-κB和MAPKs通路中的相关因子基因转录变化进行检测;然后选用NF-κB和MAPKs通路中的4种特异性抑制剂(即NF-κB通路特异性抑制剂PDTC、P38通路特异性抑制剂SB202190、ERK 1/2通路特异性抑制剂PD98059、JNK通路特异性抑制剂SP600125)通过单独或相互组合处理细胞后再进行诱导培养,同时采用RT-qPCR的方法检测用抑制剂处理绵羊RECs后SBD-1mRNA的转录水平。结果表明:酿酒酵母菌刺激RECs后,NF-κB和MAPKs通路中各因子NF-κB、P38、JNK、ERK1/2、细胞膜受体TLR2与信号衔接蛋白MyD88的mRNA水平与未刺激组相比均有所升高,且呈显著性差异(P0.01或P0.05);通过单独或组合添加抑制剂后再诱导,均发现特异性抑制剂PDTC、SB202190、SP600125、PD98059可极显著抑制酿酒酵母菌对RECs SBD-1的上调作用(P0.01),且P38通路特异性抑制剂SB202190的抑制效果最明显。结果提示,酿酒酵母菌诱导绵羊RECs SBD-1的转录可能与TLR2-MyD88-NF-κB/MAPKs通路有关,但以TLR2-MyD88-MAPKs中的TLR2-MyD88-P38通路为主要的信号通路。
[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.
【作者单位】：内蒙古农业大学兽医学院;农业部动物疾病临床诊疗技术重点实验室;
【基金】：国家自然科学基金(31160491;31560682)
【分类号】：S826

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