酿酒酵母菌对绵羊瘤胃上皮细胞β-防御素-1(SBD-1)基因表达的影响

Effects of Saccharomyces cerevisiae on the Expression of SBD-1 in Cultured Ruminal Epithelial Cells of Sheep

【目的】探索益生性酿酒酵母菌对绵羊瘤胃上皮细胞β-防御素-1(sheep beta-defensin-1,SBD-1)表达的调节作用,为在分子水平上揭示益生菌对防御素的调控机理提供一定的理论基础及依据。【方法】首先将新鲜的绵羊瘤胃组织用PBS和抗生素处理后进行瘤胃上皮细胞原代培养,当原代细胞数量长到细胞培养瓶的85%以上时,将细胞传于12孔板用于细胞刺激试验。同时将活化并纯化后的酿酒酵母菌25℃、180 r/min有氧培养48 h后,进行5次平行平板计数试验,根据平板计数的数据处理方法,得到浓度为5.2×108CFU·m L-1的酿酒酵母菌液,再通过倍比稀释把所得菌液依次稀释成浓度为5.2×107、5.2×106、5.2×105、5.2×104CFU·m L-1的菌液,用以上不同浓度(5.2×104—5.2×108CFU·m L-1)的酿酒酵母菌对体外培养的绵羊瘤胃上皮细胞分别进行不同时间(2、4、8、12、24 h)的刺激,并设置对照组用DMEM/F12培养基代替酿酒酵母菌。然后提取总RNA,逆转录为c DNA后,利用实时荧光定量PCR技术和酶联免疫吸附测定(ELISA)法检测瘤胃上皮细胞中SBD-1表达差异。最后用SAS 9.2软件对数据进行相关差异性分析。【结果】荧光定量PCR结果表明,在各时间组内SBD-1 m RNA的表达量随着菌液浓度的增加均呈先升高后降低的趋势,当菌液浓度为5.2×107CFU·m L-1时表达量达到最大,并与对照组和其他浓度组相比差异极显著(P<0.01)。当菌液浓度一定时,SBD-1 m RNA的表达量先是随着刺激时间的延长而呈上升趋势,刺激时间为12 h时SBD-1 m RNA表达量达到峰值,之后呈下降趋势。因此,当浓度为5.2×107CFU·m L-1的菌液刺激瘤胃上皮细胞12 h后,SBD-1基因的表达量达到最高,且与此浓度下其他时间组的表达量相比差异极显著(P<0.01)。ELISA检测结果显示,SBD-1蛋白表达趋势与SBD-1 m RNA检测结果基本一致。不同浓度的菌液分别刺激绵羊瘤胃上皮细胞2、4、8、12、24 h后均能提高共培养上清中SBD-1蛋白的表达,且与对照组相比存在极显著差异(P<0.01)。在菌液浓度为5.2×107CFU·m L-1刺激瘤胃上皮细胞12 h后SBD-1蛋白分泌水平达到最高,与此浓度下各时间组相比差异极显著(P<0.01)。【结论】酿酒酵母菌能够提高绵羊瘤胃上皮细胞SBD-1的表达,且菌液浓度为5.2×107CFU·m L-1刺激瘤胃上皮细胞12 h后,SBD-1的表达量达到最高。

【Objective】 The regulatory effect of probiotic Saccharomyces cerevisiae on the β-defensin-1（Sheep Beta-Defensin-1, SBD-1） expression was explored in cultured ruminal epithelial cells of sheep, which would provide a theoretical foundation and basis for the regulation mechanism of probiotics to defensins at the molecular level. 【Method】 Firstly, the primary culture of the sheep ruminal epithelial cells were carried out after treating the sheep ruminal tissue with PBS and antibiotic. When the number of cells in primary cell culture bottles grew to more than 85%, the cells were passaged to 12-well plates for cell stimulation test, meanwhile, a parallel plate count test was conducted for five times after the activated and purified S. cerevisiae were cultured at 25℃, 180 r/min aerobically for 48 h. A concentration of 5.2 × 10^8 CFU·m L^-1 S. cerevisiae was obtained according to processing method of plate count, then the concentration was further diluted to the concentrations of 5.2×10^7, 5.2×10^6, 5.2×10^5 and 5.2×10^4CFU·m L^-1 by a serial dilution. The cultured sheep ruminal epithelial cells were stimulated for different times（2, 4, 8, 12 and 24 h） by different concentrations of S. cerevisiae（5.2×10^4- 5.2×10^8 CFU·m L^-1）, and a control group was set up with DMEM/F12 medium instead of S. cerevisiae. Secondly, the total RNA was extracted from the tested cells and reversely transcripted into c DNA, then different expressions of SBD-1 were examined by real time fluorescence quantitative PCR and enzyme-linked immunosorbent assay（ELISA） tests. Finally, the related differences analysis of data was done by SAS 9.2 software. 【Result】The realtime fluorescence quantitative PCR results showed that the expression of SBD-1 m RNA in each time group increased and then decreased following the increase of concentrations, the expression of SBD-1 m RNA was the highest when the cells were stimulated with the concentration of 5.2 × 10^7 CFU·m L^-1, and significantly higher than the control group and the other concentration groups（P〈0.01）. When the concentration was constant, the expression of SBD-1 m RNA increased with the extension of stimulating time, which the expression of SBD-1 m RNA was the highest at 12 h, then downward. Therefore, the expression of SBD-1 was the highest at the concentration of 5.2 × 10^7 CFU·m L^-1 and 12 h stimulating, compared with the other time groups at this concentration was significantly different（P〈0.01）. The results of ELISA test showed that the expression trend of SBD-1 protein was consistent with the SBD-1 m RNA. After the sheep ruminal epithelial cells were stimulated with different concentrations of S. cerevisiae for 2, 4, 8, 12 and 24 h, the expression of SBD-1 protein in the co-culture supernatants increased, and there was a significant difference compared with the control group（P〈0.01）. The expression of SBD-1 protein reached the highest level at concentration of 5.2 × 10^7 CFU·m L^-1 and 12 h stimulating, the difference was significant compared with the each time group under this concentration（P〈0.01）.【Conclusion】The results of this study indicated that S. cerevisiae could improve the expression of SBD-1 in ruminal epithelial cells of sheep. And the level of SBD-1 expression was the highest when the ruminal epithelial cells were stimulated for 12 h with the concentration of 5.2 × 10^7 CFU·m L^-1.