酿酒酵母菌诱导绵羊防御素SBD-1表达的有效成分及其激活的信号通路研究

The model of indoor sheep production is a demand for return grazing land to grassland, however, breeding indoor not only increase the cost of breeding and decrease the utilization of forage, but also increase the risk of infecting diseases. Therefore enhancing the study on improving the utilization of forage and ability of resistance to disease will be very important. Defensins are a kind of endogenous antimicrobial peptide produced by animals. Probiotics not only promote the digestion and absorption, but also induce the expression of defensin. Yet the active ingredients and mechanisms which probiotics induce defensin expression are not completely known.It has been testified by our team that Saccharomyces Cerevisiae (SC) can improve the expression of the sheep β-defensin-1 (SBD-1) expression in cultured ruminal epithelium of sheep. In order to confirm the active ingredients of SC for inducing defensin, the project will study the expressive influence of extracted the ingredients to SBD-1 from the mRNA and protein level using the methods of realtime fluorescence quantitative PCR and enzyme-linked immunosorbent assay (ELISA) test through putting the different extracted ingredients in cultured ruminal epithelium of sheep. After ascertaining the active ingredients of SC, the signalling pathways of the ingredients activating the SBD-1 expression will be studied so that the mechanisms of SC regulating SBD-1 will be revealed from the molecular level. The study results will have importance for understanding the relationship between probiotics and defensins, and have practical significance for developing better the yeast to feed probiotics products to indoor sheep.

舍饲养羊模式是退牧还草工程的要求，但舍饲不仅增加养殖成本，降低饲料利用率，还增加感染疾病的风险。因此，加强对舍饲羊饲料利用率和抗病能力提高的研究意义重大。防御素是动物体内产生的一类内源性抗菌肽，益生菌不仅可以促进消化吸收，还可诱导防御素表达增加。而关于益生菌诱导防御素表达的有效成分及机理尚不完全清楚。本课题组已证明酿酒酵母菌能诱导绵羊瘤胃上皮细胞内防御素SBD-1表达增多。本项目拟提取酿酒酵母菌成分，在体外培养的绵羊瘤胃上皮细胞内加入提取的不同成分，采用荧光定量PCR和ELISA等方法，从mRNA和蛋白质水平研究酿酒酵母菌不同成分对SBD-1表达的影响，以确定有效诱导成分，并研究有效成分激活防御素表达的信号通路，从分子水平上揭示酿酒酵母菌对绵羊防御素的调控机理。该研究成果对理解益生菌与防御素之间的关系有重要意义，也为更好的开发利用酵母菌对舍饲羊进行人工饲喂益生菌制剂具有重要的实践指导意义。

为了阐明酿酒酵母菌诱导绵羊瘤胃上皮细胞（Ovine ruminal epithelial cells，ORECs）内β-防御素-1（Sheep β-defensin-1，SBD-1）的有效成分及机理，本项目首先对ORECs冻存和复苏进行研究。然后选用酿酒酵母菌细胞壁及其细胞壁蛋白、甘露聚糖和β-葡聚糖以及酿酒酵母培养物和提取物，与体外培养的ORECs共培养，通过qPCR和ELISA检测ORECs内SBD-1的表达。然后对主要诱导成分酿酒酵母细胞壁、甘露聚糖和β-葡聚糖进行诱导机制的研究。另外，还对绵羊瘤胃外植体（OREs）的体外培养及诱导表达进行了研究。.结果表明（1）成功冻存和复苏ORECs，并确定了最佳冻存液的配制比例为VFBS︰VDMEM︰VDMSO = 9︰0︰1，大大节省了研究的材料和时间。（2）酿酒酵母菌各成分对ORECs的诱导表达均与诱导物的浓度和诱导时间具有依赖性，但最佳诱导浓度和诱导时间不同。酿酒酵母细胞壁浓度为200μg/mL诱导培养12h时ORECs SBD-1表达最高；酿酒酵母菌细胞壁蛋白粗提物、甘露聚糖和β-葡聚糖的最佳诱导浓度和时间分别为100μg/mL 12h、50μg/mL 4h和10μg/mL 2及4h；酿酒酵母培养物和提取物的最佳诱导浓度为400和200μg/mL，最佳诱导时间均为6h。说明酿酒酵母各成分均能诱导ORECs SBD-1的表达。（3）分别用酿酒酵母培养物、提取物和细胞壁各自最佳刺激浓度和时间诱导培养ORECs，结果表明酿酒酵母细胞壁刺激组SBD-1表达量最高，显著高于对照组、酿酒酵母培养物和提取物刺激组。表明酿酒酵母细胞壁诱导效果优于酿酒酵母提取物和培养物。（4）阐明酿酒酵母菌细胞壁及其成分甘露聚糖和β-葡聚糖均能激活NF-κB和MAPK信号通路，但各有侧重，酿酒酵母细胞壁主要通过TLR2—MyD88—NF-κB、甘露聚糖通过Dectin-2—Syk—MAPK（p38）、β-葡聚糖通过Dectin-1—Syk/TLR-2—MyD88—NF-κB信号通路激活ORECs SBD-1的表达。（5）确定了不添加血清的培养基、培养24h OREs组织结构较完整，并证明酿酒酵母菌和β-葡聚糖均可诱导OREs中SBD-1的表达。本研究结果为绵羊进行人工饲喂益生菌制剂具有重要的实践指导意义。