脾酪氨酸激酶参与酿酒酵母甘露聚糖诱导绵羊瘤胃上皮细胞β-防御素-1(SBD-1)的表达

doi:10.11843/j.issn.0366-6964.2019.05.019

摘要/Abstract

摘要：

为了探索脾酪氨酸激酶（Syk）是否参与酿酒酵母甘露聚糖（S.c M）诱导绵羊瘤胃上皮细胞（ORECs）β-防御素-1（SBD-1）表达的过程，首先利用免疫组化、RT-PCR和免疫荧光等方法检测Syk在ORECs内的表达情况；然后采用qPCR和Western blot方法检测S.c M刺激ORECs后Syk的表达变化，同时用Western blot方法检测Syk的磷酸化水平；接着用3条Syk特异性siRNAs（#1、#2和#3）转染ORECs 24 h后，qPCR检测Syk mRNA的表达变化，筛选出干扰效果最佳的Syk siRNA；最后用效果最佳的siRNA和Syk特异性抑制剂R406分别处理ORECs后，采用qPCR和ELISA检测SBD-1的表达变化，以确定Syk在S.c M诱导SBD-1表达过程中的作用。结果显示：Syk在ORECs内表达；且S.c M刺激ORECs后Syk的mRNA和蛋白表达水平显著高于未刺激组（P<0.01或P<0.05），S.c M刺激ORECs不同时间（5、15、30、45和60 min）均能使Syk发生磷酸化，且刺激15 min后磷酸化水平达到最大（P<0.01）；此外，Syk的3条特异性siRNAs转染ORECs后Syk的表达均降低，且Syk siRNA#2的抑制效果最明显（P<0.01）；同时Syk siRNA#2和R406均能极显著降低S.c M诱导ORECs SBD-1的表达（P<0.01）。上述结果表明，Syk参与S.c M诱导ORECs SBD-1的表达。

Abstract:

The aim of this study was to explore whether spleen tyrosine kinase (Syk) is involved in the expression of β-defensin-1 (SBD-1) in ovine ruminal epithelial cells (ORECs) induced by Saccharomyces cerevisiae Mannan (S.c M). Firstly, the expression of Syk in rumen tissues and ORECs was detected by immunohistochemistry, RT-PCR and immunofluorescence. Then, qPCR and Western blot were used to detect the expression of Syk after S.c M stimulation of ORECs, and the phosphorylation level of Syk was detected by Western blot. The ORECs were transfected with three Syk-specific siRNAs (#1, #2 and #3) for 24 h, qPCR was used to detect the expression of Syk mRNA, and the Syk siRNA with the best interference effect was screened. Finally, after treatment of ORECs with the best siRNA and Syk specific inhibitor R406, the expression of SBD-1 was detected by qPCR and ELISA to determine the role of Syk in the expression of SBD-1 induced by S.c M. The results showed that Syk was expressed in ORECs, and the expression level of Syk mRNA and protein were significantly higher in mannan-stimulated ORECs than in unstimulated group (P<0.05). After S.c M stimulation of ORECs for different time (5, 15, 30, 45 and 60 min), Syk was phosphorylated, and the phosphorylation level reached the maximum after 15 min stimulation (P<0.01). In addition, the expression of Syk was decreased after Syk's three specific siRNAs transfected with ORECs, and the inhibitory effect of Syk siRNA#2 was the most significant (P<0.01). Simultaneously, Syk siRNA#2 and R406 significantly reduced S.c M-induced ORECs. Expression of SBD-1 (P<0.01). The above results indicate that Syk is involved in the expression of SBD-1 induced by S.c M.

中图分类号:

S852.4

金鑫, 张曼, 杨银凤. 脾酪氨酸激酶参与酿酒酵母甘露聚糖诱导绵羊瘤胃上皮细胞β-防御素-1(SBD-1)的表达[J]. 畜牧兽医学报, 2019, 50(5): 1082-1090.

JIN Xin, ZHANG Man, YANG Yinfeng. Syk is Involved in the Expression of SBD-1 in Ovine Ruminal Epithelial Cells Induced by Saccharomyces cerevisiae Mannan[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(5): 1082-1090.

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