不同信号通路抑制剂对酿酒酵母菌诱导绵羊瘤胃上皮细胞SBD-1表达的影响  

Effect of different signaling pathway inhibitors on SBD-1 expression in sheep rumen epithelial cells induced by Saccharomyces cerevisiae

作　　者：金鑫[1,2] 张曼[1,2] 范燕茹[1,2] 王佩[1,2] 刘骄[1,2] 杨银凤[1,2] JIN Xin ZHANG Man FAN Yan-ru WANG Pei LIU Jiao YANG Yin-feng(Col lege of Veterinary Medicine, InnerMongolia Agricultural University, Hohhot 010018 ,China Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease ,Ministry of Agri culture, Hohhot 010018, China)

机构地区：[1]内蒙古农业大学兽医学院,内蒙古呼和浩特010018 [2]农业部动物疾病临床诊疗技术重点实验室,内蒙古呼和浩特010018

出　　处：《中国兽医学报》2016年第12期2067-2073,共7页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31160491;31560682)

摘　　要：为了探索核因子-kB(nuclear factor-k-gene binding,NF-kB)和丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)通路是否介导益生性酿酒酵母菌(Saccharomyces cerevisiae,SC)诱导绵羊瘤胃上皮细胞β-防御素-1(sheep beta-defensin-1,SBD-1)基因表达的调控机制。首先建立绵羊瘤胃上皮细胞培养体系作为体外试验模型,将传代细胞分为4个试验组(SC+PDTC、SC+PD98059、SC+SB202190、SC+SP600125)、4个阴性对照组(PDTC、PD98059、SB202190、SP600125)、1个阳性对照组(SC)和1个空白对照组。向试验组和阴性对照组内分别加入NFkB通路特异性抑制剂PDTC(50μmol/L)、MAPKs通路ERK 1/2特异性抑制剂PD98059(20μmol/L)、p38特异性抑制剂SB202190(20μmol/L)和JNK特异性抑制剂SP600125(20μmol/L)4种抑制剂,作用1h后,用PBS将各组细胞清洗3次,然后向试验组和阳性对照组中分别添加浓度为5.2×107 CFU/mL的酿酒酵母菌液100μL,并向阴性对照组和空白对照组中分别添加等量的DMEM/F12培养液。将以上10组细胞置于37℃、5%CO2培养箱中培养12h后提取各组细胞总RNA,利用实时荧光定量PCR技术检测各组SBD-1 mRNA表达水平的变化。结果显示:与阳性对照组相比,4种抑制剂对酿酒酵母菌促进瘤胃上皮细胞SBD-1的诱导表达都具有抑制作用,且存在极显著性差异(P<0.01),而各阴性对照组与空白对照组相比差异均不显著(P>0.05);4种抑制剂的抑制效果依次表现为SB202190>PDTC>SP600125>PD98059,且SB202190抑制效果极显著高于PDTC(P<0.01),而PDTC抑制效果与SP600125、PD98059相比差异均不显著(P>0.05)。结果表明,NF-kB和MAPKs通路可能均介导酿酒酵母菌诱导绵羊瘤胃上皮细胞SBD-1的表达,但可能以MAPKs途径为主要信号通路。In order to explore the regulation mechanism of probiotic Saccharomyces cerevisiae indu- cing sheep beta-defensin 1 （SBD-1） gene expression in sheep rumen epithelial cells,we studied the NF-kB and MAPKs signal pathways. Firstly, the ruminal epithelium cells of sheep were cultured successfully as experimental model in vitro, and the passaged cells were divided into four experi- mental groups （SC＋PDTC, SC＋PD98059, SC＋ SB202190, SC＋ SP600125）, four negative control group （PDTC,PD98059 ,SB202190,SP600125） ,a positive control group （SC） and a control group. Secondly,added four kinds of inhibitors of NF-kB pathway inhibitor PDTC （50 μmol/L）,MAPKs pathway ERK 1/2 specific inhibitor PD98059 （20 μmol/L）, p38 specific inhibitor SB202190 （20μmol/L）,JNK specific inhibitor SP600125 （20 μmol/L） into the experimental group and the negative control group,after treatment 1 h, each group cells were washed three times with PBS, then 100μL of Saccharomyces cerevisiae at the concentration of 5.2×10 7 CFU/mL was added to the experimental group and the positive control group,and added an equal volume of DMEM/F12 medium to the negative control group and control group. Finally, cells of ten groups were incuba- toredat 37℃,5% CO2 for 12 h,then total RNA was extracted from each group,the changes of SBD-1 mRNA expression levels were examined by real-time fluorescence quantitative PCR. The results showed that four kinds of inhibitors can significantly inhibite the SBD-1 expression in ru- men epithelial cells induced by Saccharomyces cerevisiae （P〈0. 01）, compared with the positive control group,but each negative control group was no significant difference （P〉0.05） compared to the control group. The inhibitory effect of four kinds of inhibitors was followed by SB202190） PDTC〉SP600125〉PD98059,and the inhibitory effect of SB202190 was significantly higher than PDTC （P〈0.01）,while the inhibitory effect of PDTC was no significant difference （P〉0.05） compared with SP600

关 键 词：β-防御素-1 瘤胃上皮细胞 酿酒酵母菌 抑制剂 信号通路 实时荧光定量PCR 

分 类 号：S852.4[农业科学—基础兽医学] S852.6[农业科学—兽医学]