酿酒酵母β-葡聚糖通过膜受体Dectin-1和TLR-2诱导绵羊瘤胃上皮细胞表达SBD-1的机制研究

Mechanism of Saccharomyces Cerevisiae β-glucan Inducing SBD-1 Expression in Ovine Rumen Epithelial Cells by Membrane Receptors Dectin-1 and TLR-2

为了探索酿酒酵母β-葡聚糖诱导绵羊瘤胃上皮细胞(ovine ruminal epithelial cells,ORECs)β-防御素-1(sheepβ-defensin-1,SBD-1)表达过程中膜受体Dectin-1和TLR-2可能存在的作用。本研究首先利用免疫组化、RT-PCR、免疫荧光和Western blot等方法检测Dectin-1是否在ORECs内表达,并且采用qPCR和Western blot方法对β-葡聚糖刺激ORECs后细胞膜受体Dectin-1和TLR-2的表达变化进行检测。而后用不同浓度的Dectin-1阻断剂昆布多糖或TLR-2特异性封闭抗体分别预处理ORECs后,采用qPCR和ELISA方法检测SBD-1的表达变化,以确定β-葡聚糖诱导SBD-1表达过程中Dectin-1和TLR-2的参与情况。结果显示:1)绵羊瘤胃组织及ORECs内存在Dectin-1表达,且β-葡聚糖刺激ORECs后Dectin-1和TLR-2的表达水平显著增加(P<0.05);2)不同浓度的昆布多糖和TLR-2封闭抗体均可以极显著降低β-葡聚糖诱导SBD-1的表达(P<0.01),且随着阻断剂和封闭抗体浓度的增加,其抑制SBD-1表达的作用越明显。结果表明,Dectin-1在绵羊瘤胃组织及ORECs内均表达,并且酿酒酵母β-葡聚糖在ORECs中诱导SBD-1的表达是由Dectin-1和TLR-2介导产生的。

The aim of this study was to explore the possible role of TLR-2 and Dectin-1 in the expression ofβ-defensin-1(SBD-1)in ovine ruminal epithelial cells(ORECs)induced by Saccharomyces cerevisiaeβ-glucan.The immunohistochemistry,RT-PCR,immunofluorescence and Western blot were used to detect the expression of Dectin-1 in ORECs,and qPCR and Western blot were used to detected the expression changes of Dectin-1 and TLR-2 afterβ-glucan stimulated ORECs.Then,ORECs were pretreated using Dectin-1 blocker laminarin or TLR-2 blocking antibody with different concentrations,and the expression of SBD-1 were detected by qPCR and ELISA to determine the involvement of Dectin-1 and TLR-2 in the induction expression of SBD-1 induced byβ-glucan.The results showed that:1)Dectin-1 was expressed in ovine ruminal tissues and ORECs,and the expression levels of Dectin-1 and TLR-2 were significantly increased afterβ-glucan stimulating ORECs(P<0.05);2)The laminarin or TLR-2 blocking antibody with different concentrations significantly decreased the expression of SBD-1 induced byβ-glucan(P<0.01),and this effect became stronger as the laminarin and blocking antibodies concentration increased.The results indicate that Dectin-1 is expressed in both ovine ruminal tissues and ORECs,and the Saccharomyces cerevisiaeβ-glucan inducing SBD-1 expression in ORECs is mediated by Dectin-1 and TLR-2.