酿酒酵母β-葡聚糖对绵羊瘤胃上皮细胞β-防御素-1表达的影响  

Effects of Saccharomyces cerevisiae β-glucan on the Expression of SBD-1 in Ovine Ruminal Epithelial Cells

作　　者：张曼[1,2] 金鑫 王云鹤[1,2] 魏方 温婧怡[1,2] 李政忆 杨银凤 ZHANG Man;JIN Xin;WANG Yun he;WEI Fang;WEN Jing yi;LI Zheng yi;YANG Yin feng(College of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot 010018,China;Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease of Ministry of Agriculture,Hohhot 010018,China)

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

出　　处：《畜牧兽医学报》2018年第11期2416-2424,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金项目(31560682)

摘　　要：为了探索β-葡聚糖对绵羊瘤胃上皮细胞(ruminal epithelial cells,RECs)β-防御素-1(sheepβ-defensin-1,SBD-1)表达的影响。本研究首先建立绵羊RECs培养体系作为体外试验模型,用不同浓度(0、5、10、20、50和100μg·mL^(-1))的β-葡聚糖刺激RECs 8h后,利用qPCR和ELISA方法检测RECs中SBD-1的表达变化,选出诱导SBD-1表达最高的β-葡聚糖浓度。然后用β-葡聚糖的最佳刺激浓度对RECs分别刺激0、2、4、8、12和24h,同样利用qPCR和ELISA方法对RECs SBD-1的表达变化进行检测,从而筛选出诱导SBD-1表达最高的刺激时间。qPCR和ELISA结果显示,β-葡聚糖(5～100μg·mL^(-1))刺激RECs 8h后,SBD-1mRNA和蛋白的表达随着β-葡聚糖浓度的升高均呈现先升高后降低趋势,且当β-葡聚糖浓度为10μg·mL^(-1)时SBD-1mRNA和蛋白的表达量极显著高于对照组(P<0.01)。当用10μg·mL^(-1)的β-葡聚糖刺激RECs 0～24h后,qPCR结果显示,10μg·mL^(-1)的β-葡聚糖刺激RECs 2h时SBD-1mRNA的表达量最高(P<0.01),之后呈下降趋势,而ELISA结果显示,在β-葡聚糖浓度为10μg·mL^(-1)刺激RECs 4h后SBD-1蛋白分泌水平达到最高(P<0.01)。MTT测定结果显示,100μg·mL^(-1)β-葡聚糖会对绵羊RECs活力产生显著的影响(P<0.05)。本研究结果表明,β-葡聚糖能够提高绵羊瘤胃上皮细胞SBD-1的表达,且用浓度为10μg·mL^(-1)的β-葡聚糖分别刺激RECs 2和4h后SBD-1的mRNA和蛋白表达达到最高。The aim of this study was to explore the effect of Saccharomyces cerevisiae β-glucan on the β-defensin-（SBD-1） expression in cultured ruminal epithelial cells（RECs） of ovine. Firstly, the ovine RECs culture system was established as an in vitro experimental model, the RECs were stimulated with various concentrations （0, 5, 10, 20, 50 and 100 μg·mL 1） of glucanfor 8 h, and the expression level of SBD 1 mRNA and protein in RECs were detected by qPCR and ELISA to determine the concentration of β-glucan that induced the highest expression of SBD 1. Then, RECs were stimulated with the optimal stimulating concentration of β-glucan for 0, 2, 4, 8, 12 and 24 h, respectively and the expression of RECs SBD 1 were also detected by qPCR and ELISA, so as to determine the optimal stimulation time for β-glucan induced SBD 1 expression. The qPCR and ELISA results showed that after β-glucan （5 100 μg · mL 1） stimulated RECs for 8 h, the expression of SBD 1 mRNA and protein were increased and then decreased following the increase of concentrations , and when the concentration of β-glucanwas 10μg·mL 1, theexpression level of SBD 1 mRNA and protein were significantly higher than the control group （P〈0.01）. When RECs were stimulated with 10 μg · mL β-glucan for 0 24 h, qPCR results showedthat SBD 1 mRNA expression was the highest at 2 h （P〈0.01）, and then downward; The results of ELISA test showed that SBD 1 protein secretion reached the highest level at 4 h （P〈0.01）. The results of MTT test showed that 100 μg·mL β-glucan significantly affect the sheepRECs viability （P〈0.05）. The results of this study indicated that β-glucan could improve theSBD 1 expression in RECs of ovine. And the level of SBD 1 mRNA and protein expression wasthe highest when the RECs were separately stimulated for 2 and 4 h with the 10μg·mL 1 β-glucan.

关 键 词：β-防御素-1 瘤胃上皮细胞 Β-葡聚糖 qPCR ELISA MTT 

分 类 号：S826.2[农业科学—畜牧学]