短链脂肪酸蓄积抑制SLC5A8和MCT1表达并加重亚急性瘤胃酸中毒

发布时间：2018-05-08 17:41

本文选题：亚急性瘤胃酸中毒 + 钠离子耦合单羧酸转运蛋白1　；参考：《吉林大学》2017年硕士论文

【摘要】：亚急性瘤胃酸中毒(SARA)是公认的高产奶牛消化系统性疾病,因其危害奶牛健康和生产性能而受到重视。在规模化养殖条件下,为满足高产奶牛对能量的需求,过度饲喂精饲料,使短链脂肪酸(SCFA)在瘤胃内的产生速率超过吸收速率,从而引起SCFA在瘤胃内大量蓄积,导致瘤胃p H下降,并增加SARA发生的风险。SCFA是反刍动物重要的能量代谢基底物,其在机体内代谢产生大量的能量,因此,研究SCFA在瘤胃内的转运和吸收机制,对反刍动物能量供应具有至关重要的作用,然而,目前SCFA在瘤胃内的转运和吸收机制尚不清楚。在人和鼠的肠上皮细胞,钠离子耦合单羧酸转运蛋白1(SLC5A8)和氢离子耦合单羧酸转运蛋白1(MCT1)对SCFA的吸收和转运至关重要,然而对于奶牛SLC5A8和MCT1的研究却鲜有报道。本研究通过体内实验和体外实验相结合的方法对SLC5A8和MCT1进行了研究。在体内试验(1)通过Western blot和实时荧光定量PCR检测SLC5A8和MCT1在犊牛和成年牛消化道内的分布和表达,结果表明,SLC5A8和MCT1在犊牛和成年牛的消化道内均有表达和分布,其中瘤胃的表达水平显著高于其它部位,且SLC5A8和MCT1在成年牛的大肠中的表达显著升高;(2)为了确定SLC5A8和MCT1的表达变化与奶牛健康的关系,检测了SLC5A8和MCT1在正常奶牛和SARA奶牛瘤胃内的表达变化,结果显示,SARA奶牛抑制了SLC5A8和MCT1的表达;(3)通过免疫组织化学的方法检测SLC5A8和MCT1在瘤胃组织中的定位,发现SLC5A8主要分布于近管腔侧的瘤胃上皮组织中,MCT1主要位于瘤胃上皮的基底层。为了确定SLC5A8和MCT1的表达在SARA奶牛被抑制的原因,体外分离培养犊牛瘤胃上皮细胞,根据正常和SARA奶牛瘤胃内SCFA的浓度,将SCFA分为SARA组(乙酸90 m M、丙酸40 m M和丁酸30 m M)和对照组(乙酸60 m M,丙酸30 m M和丁酸20 m M)处理瘤胃上皮细胞,发现低浓度的SCFA促进SLC5A8和MCT1的表达,高浓度的SCFA抑制SLC5A8和MCT1的表达,这与瘤胃体内的结果一致。为了进一步确定影响SLC5A8和MCT1表达的具体因素,用不同浓度的乙酸(0,30,60和90 m M)、丙酸(0,20,30和40 m M)和丁酸(0,10,20和30 m M)处理瘤胃上皮细胞,结果显示,低浓度的丙酸和丁酸促进SLC5A8和MCT1的表达,高浓度的丙酸和丁酸抑制SLC5A8和MCT1的表达;乙酸对SLC5A8的表达具有促进作用,并随浓度的升高而逐渐上升,然而高浓度的乙酸抑制了MCT1的表达。另外,通过瘤胃上皮细胞免疫荧光实验,发现SLC5A8和MCT1主要位于细胞膜的表面,其主要通过跨膜的方式进行转运。综上所述,这些结果表明高浓度的SCFA抑制SLC5A8在SARA奶牛的瘤胃中的表达,降低SCFA的吸收,从而加重SARA。
[Abstract]:Subacute rumen acidosis (SARAA) is a widely recognized digestive system disease in dairy cows. In order to meet the energy demand of high-yield dairy cattle, the short chain fatty acid (SCFA) production rate in rumen exceeded the absorption rate in order to satisfy the energy demand of high-yield dairy cows, which led to the accumulation of SCFA in rumen. SARA is an important energy metabolic substrate in ruminants, which produces a large amount of energy in the body. Therefore, the mechanism of SCFA transport and absorption in rumen is studied. However, the mechanism of SCFA transport and absorption in rumen is still unclear. In human and mouse intestinal epithelial cells, sodium ion-coupled monocarboxylate transporter (1SLC5A8) and hydrogen ion-coupled monocarboxylic acid transporter (1MCT1) play an important role in the absorption and transport of SCFA. However, there are few studies on SLC5A8 and MCT1 in dairy cattle. In this study, SLC5A8 and MCT1 were studied by in vivo and in vitro experiments. The distribution and expression of SLC5A8 and MCT1 in the digestive tract of calves and adult cattle were detected by Western blot and real-time fluorescence quantitative PCR. The results showed that SLC5A8 and MCT1 were expressed and distributed in the digestive tract of calves and adult cattle. In order to determine the relationship between the expression of SLC5A8 and MCT1 and the health of cows, the expression of SLC5A8 and MCT1 in the large intestine of adult cattle was significantly higher than that in other parts. The expression of SLC5A8 and MCT1 in the rumen of normal cows and SARA cows were detected. The results showed that the expression of SLC5A8 and MCT1 was inhibited in Sara cows. The localization of SLC5A8 and MCT1 in rumen tissues was detected by immunohistochemical method. It was found that SLC5A8 was mainly located in the basal layer of rumen epithelium in the proximal lumen of rumen epithelium. In order to determine the reason for the inhibition of SLC5A8 and MCT1 expression in SARA cows, calf rumen epithelial cells were isolated and cultured in vitro, and the concentrations of SCFA in the rumen of normal and SARA cows were determined. SCFA was divided into SARA group (acetic acid 90 mm, propionic acid 40 mm and butyric acid 30 mm) and control group (acetic acid 60 mm, propionic acid 30 mm and butyric acid 20 mm). It was found that low concentration of SCFA promoted the expression of SLC5A8 and MCT1. High concentration of SCFA inhibits the expression of SLC5A8 and MCT1, which is consistent with the results in rumen. In order to further determine the specific factors that affect the expression of SLC5A8 and MCT1, the rumen epithelial cells were treated with different concentrations of 30 and 90 mg / L acetate, 2030 and 40 mm propionate, and 1020 and 30 mm butyrate, respectively. Low concentration of propionic acid and butyric acid promoted the expression of SLC5A8 and MCT1, high concentration of propionic acid and butyric acid inhibited the expression of SLC5A8 and MCT1, acetic acid promoted the expression of SLC5A8 and increased gradually with the increase of concentration. However, high concentration of acetic acid inhibited the expression of MCT1. In addition, it was found that SLC5A8 and MCT1 were mainly located on the surface of the cell membrane and transported mainly through the membrane through the immunofluorescence assay of rumen epithelial cells. In conclusion, these results suggest that high concentration of SCFA inhibits the expression of SLC5A8 in the rumen of SARA cows and decreases the absorption of SCFA, thus exacerbating SARAA.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.23

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