【摘要】 为了探索枯草芽孢杆菌细胞壁诱导绵羊β-防御素-1(SBD-1)表达的主要信号转导途径，本试验首先使用反复冻融法与超声波破碎法相结合的方法破碎枯草芽孢杆菌并收集其细胞壁，酶联免疫吸附测定(ELISA)检测方法分析细胞壁成分及其含量，然后利用实时荧光定量PCR(RT-qPCR)和蛋白质免疫印迹(Western blot, WB)方法检测枯草芽孢杆菌细胞壁刺激绵羊瘤胃上皮细胞(ORECs)后通路因子(TLR-2、MyD88、NF-κB、p38、JNK和ERK1/2)mRNA和蛋白的表达变化，最后使用通路抑制剂分别阻断NF-κB、p38、JNK和ERK1/2信号通路，用枯草芽孢杆菌细胞壁刺激ORECs并检测刺激后SBD-1 mRNA表达变化，从而确定诱导SBD-1表达的主要信号通路。结果显示，枯草芽孢杆菌细胞壁制备成功，细胞壁主要成分为肽聚糖和磷壁酸，含量分别为(90.85±0.91)%和(8.85±0.11)%。与对照组相比，枯草芽孢杆菌细胞壁刺激ORECs后信号通路因子(TLR-2、MyD88、NF-κB、p38、JNK和ERK1/2)的mRNA和蛋白的表达水平均显著升高(P<0.05或P<0.01),使用通路抑制剂阻断各个信号通路后再刺激ORECs,SBD-1 mRNA表达水平均极显著下降(P<0.01),且添加NF-κB和JNK抑制剂组下降幅度最大。结果表明，枯草芽孢杆菌细胞壁可能通过TLR-2-MyD88-NF-κB/MAPK信号通路调控ORECs SBD-1的表达。更多还原

【Abstract】 To explore the major signaling transduction pathway for Bacillus subtilis cell wall-induced expression of sheep β-defensin-1(SBD-1) in ovine rumen epithelial cells(ORECs), this study first used a combination of freeze-thaw and ultrasonic disruption methods to break Bacillus subtilis and collected its cell wall. Enzyme-linked immunosorbent assay(ELISA) was used to analyze the composition and content of the cell wall. Real-time quantitative PCR(RT-qPCR) and Western blot(WB) were then used to detect the changes in mRNA and protein expression of pathway factors(TLR-2, MyD88, NF-κB, p38, JNK and ERK1/2) in ORECs stimulated with Bacillus subtilis cell wall. Finally, pathway inhibitors were used to block the NF-κB, p38, JNK, and ERK1/2 signaling pathways respectively, stimulated ORECs with Bacillus subtilis cell wall, and detected the changes in SBD-1 mRNA expression after stimulation to determine the main signaling pathway for inducing SBD-1 expression. The results showed that the Bacillus subtilis cell wall was successfully prepared,with the main components being peptidoglycan and teichoic acid,accounting for( 90. 85 ±0. 91) % and( 8. 85 ± 0. 11) %,respectively. Compared with the control group,the mRNA and protein expression levels of pathway factors( TLR-2,MyD88,NF-κB,p38,JNK and ERK1/2) in ORECs significantly increased after stimulation with Bacillus subtilis cell wall( P < 0. 05 or P < 0. 01). After blocking each signaling pathway with pathway inhibitors and then stimulating ORECs,the mRNA expression level of SBD-1 significantly decreased( P < 0. 01),with the largest decrease observed in the NF-κB and JNK inhibitor groups. The results indicate that the Bacillus subtilis cell wall may regulate the expression of SBD-1 in ORECs through the TLR-2-MyD88-NF-κB/MAPK signaling pathway.更多还原