奶牛组织器官SLC15家族的表达及二肽对小肠上皮细胞SLC15家族的影响

发布时间：2018-04-11 21:40

本文选题：成年奶牛 + SLC15家族　；参考：《扬州大学》2017年硕士论文

【摘要】：质子转运载体15家族(Solute carrier 15 family,SLC15)又被称为小肽转运载体家族,主要位于细胞膜上,能够利用电化学梯度将短链肽和拟肽类物质转运进入细胞内。SLC15家族成员主要包括SLC15A1、SLC15A2、SLC15A3和SLC15A4,目前鲜有报道能够全面地研究这些小肽转运载体在成年奶牛上的表达差异。小肽转运载体能够转运肠道中的营养物质,它也受这些营养物质调控。丙氨酰谷氨酰胺二肽(alanyl-glutamine,Ala-Gln)作为小肠的主要能源物质,它对肠道中小肽转运载体的调控也鲜有研究。因此本试验研究SLC15A1、SLC15A2、SLC15A3和SLC15A4在成年奶牛组织器官中的表达情况以及Ala-Gln对奶牛小肠上皮细胞(Bovine intestinal epithelial cells,BIECs)中小肽转运载体的调控作用,为SLC15家族在成年奶牛中的生物学功能以及Ala-Gln对BIECs的调控机制提供科学依据。试验一、小肽转运载体在成年奶牛组织器官中的表达试验选取3头健康的成年荷斯坦奶牛进行屠宰,采集心脏、肝脏、脾脏、肺脏、肾脏、瘤胃、网胃、瓣胃、皱胃、十二指肠、空肠、回肠、盲肠、结肠和直肠。试验采用实时荧光定量PCR法对器官组织中的SLC15A1、SLC15A2、SLC15A3和SLC15A4 mRNA进行分析;采用Western Blot方法对器官组织中的SLC15A1、SLC15A2和SLC15A4蛋白进行分析。试验结果发现SLC15A1、SLC15A2、SLC15A3和SLC15A4mRNA在器官组织中均能表达,但是在回肠中没有发现SLC15A2 mRNA的表达。SLC15A1在成年奶牛的肺脏、肾脏和空肠的表达极显著高于其它器官组织(P0.01);SLC15A2在肾脏组织中的表达量均极显著高于其它器官组织(P0.01);SLC15A3mRNA在瘤胃、脾脏、结肠和肺脏中的表达极显著高于其它器官组织(P0.01);SLC15A4在脾脏,肾脏,肝脏,盲肠,结肠和肺脏中均有较高的表达水平。试验二、奶牛小肠上皮细胞原代培养及其小肽转运载体的表达在无菌条件下分离奶牛小肠上皮组织,用胰酶消化获得BIECs并纯化,免疫荧光和琼脂糖凝胶电泳鉴定BIECs,实时荧光定量PCR检测细胞内SLC15A1、SLC15A2、SLC15A3和 SLC15A4 mRNA 表达;Western Blot 检测细胞中 SLC15A1、SLC15A2 和 SLC15A4 蛋白表达。结果表明,BIECs的细胞质中均含有细胞角蛋白18荧光,在细胞中检测到绒毛蛋白、上皮细胞钙粘蛋白和小肠脂肪酸结合蛋白基因的表达证实细胞为纯化的BIECs。细胞中SLC15A1 mRNA 的表达量极显著高于 SLC15A2、SLC15A3 和 SLC15A4 mRNA(P0.01)。SLC15A1蛋白的表达量最高,极显著高于SLC15A2和SLC15A4(P0.01)。试验三、Ala-Gln对奶牛小肠上皮细胞中小肽转运载体表达的影响不同时间和浓度的Ala-Gln培养BIECs,确定最佳培养时间和浓度。用实时荧光定量PCR 检测 Ala-Gln、丙氨酸(L-alanine,Ala)和谷氨酰胺(L-glutamine,Gln)对 BIECs中小肽转运载体及其相关基因mRNA的影响,Western Blot检测Ala-Gln、Ala和Gln对BIECs中SLC15A1、SLC15A2和SLC15A4蛋白的影响。结果发现,Ala-Gln能够促进BIECs增殖,其中1mMAla-Gln在4h时极显著促进细胞增殖(P0.01),并且该组中SLC15A1 mRNA、SLC15A2 mRNA、SLC15A3 mRNA 和 SLC15A4 mRNA 的表达量极显著高于其它各组(P0.01),显著促进SLC15A1蛋白的表达(P0.05)。Cdx2和PPAR-α mRNA的提高能够上调SLC15A1mRNA的表达量。Ala-Gln培养BIECs后发现磷脂酰肌醇3激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶 B(protein kinase B,AKT)信号通路关键基因的表达都显著升高(P0.05)。综上,SLC15A1、SLC15A2、SLC15A3和SLC15A4在成年奶牛中广泛表达,但是它们在器官组织中的表达水平存在较大差异。SLC15A1 mRNA在BIECs中表达量最高,主要是通过Cdx2和PPAR-α基因调控的。Ala-Gln能够上调小肽转运载体的表达,并且能上调PI3K/AKT传导信号中关键基因的表达。
[Abstract]:Proton transporter 15 family (Solute carrier 15 family, SLC15) also known as the small peptide transporter family, mainly located in the cell membrane, the electrochemical gradient can use short chain peptide and peptidomimetic compounds transported into cells of.SLC15 family members including SLC15A1, SLC15A2, SLC15A3 and SLC15A4, there are few reports can be fully study on differential expression of these peptide transporters in adult dairy cows. The peptide transporter can transfer of nutrients in the gut, it is also affected by these nutrients. The regulation of alanyl glutamine peptide two (alanyl-glutamine, Ala-Gln) as the main energy source of the small intestine, study its regulation on small intestinal peptide transporter is rare therefore, this study. SLC15A1, SLC15A2, SLC15A3 and SLC15A4 expression in adult cow tissues and Ala-Gln of intestinal epithelial cells (Bovine intestina in dairy cows L epithelial cells, BIECs) Regulation of small peptide transporters, and provide scientific basis for the regulation of the biological function of SLC15 family in adult dairy cattle and Ala-Gln of BIECs. A test, small peptide transporter expression test in adult dairy cows tissues from 3 healthy adult Holstein cows were slaughtered, collecting heart the liver, spleen, lung, kidney, and rumen, reticulum, omasum, abomasum, duodenum, jejunum, ileum, cecum, colon and rectum. Test by using real-time fluorescence quantitative PCR method for SLC15A2 in organs and tissues of SLC15A1, SLC15A3, SLC15A4 and mRNA were analyzed by Western; method of Blot in organs and tissues of SLC15A1 SLC15A2 and SLC15A4 protein were analyzed. The experimental results showed that SLC15A1, SLC15A2, SLC15A3 and SLC15A4mRNA were expressed in organs and tissues, but no expression of.SL SLC15A2 mRNA in ileum C15A1 in adult cow lung, expression of kidney and jejunum were significantly higher than that of other organs (P0.01); the expression of SLC15A2 in renal tissues were significantly higher than other organs (P0.01); SLC15A3mRNA in the rumen, spleen, colon and lung expression in extremely significantly higher than that of other organs (P0.01); SLC15A4 in the spleen, kidney, liver, cecum, colon and lung were expressed at higher levels. In experiment two, expression of bovine intestinal epithelial cells in primary culture and small peptide transporter under aseptic conditions of cow intestine and skin tissue isolated, digested with trypsin and purified BIECs, immunofluorescence and BIECs agarose gel electrophoresis., real time fluorescence quantitative PCR detection of intracellular SLC15A1, SLC15A2, SLC15A3 and SLC15A4 mRNA SLC15A1 Western expression; Blot were detected, SLC15A2 and SLC15A4 protein expression. The results showed that BIECs cells With cytokeratin 18 fluorescence in cytoplasm of villus protein in cells, cells confirmed expression of purified BIECs. cells in SLC15A1 mRNA were significantly higher than that of SLC15A2 and E-cadherin in intestinal fatty acid binding protein gene expression, SLC15A3 and SLC15A4 mRNA (P0.01).SLC15A1 protein expression was the highest higher than that of SLC15A2 and SLC15A4 (P0.01). In experiment three, effect of Ala-Gln on the expression of small peptide transporter of cow intestinal epithelial cells carrier in different time and concentration of Ala-Gln in cultured BIECs, to determine the optimal incubation time and concentration. Using real-time quantitative PCR detection of Ala-Gln (L-alanine, Ala), alanine and glutamine (L-glutamine, Gln) of mRNA and the small peptide transporter gene BIECs, Western Blot and Ala Gln to detect Ala-Gln, BIECs SLC15A1, effects of SLC15A2 and SLC15A4 protein. Results, Ala-Gln can promote the proliferation of BIECs, in which 1mMAla-Gln 4H significantly promoted cell proliferation (P0.01), and the group SLC15A1 mRNA, SLC15A2 mRNA, SLC15A3 mRNA and SLC15A4 mRNA expression was significantly higher than other groups (P0.01), significantly promote the expression of SLC15A1 (P0.05).Cdx2 and PPAR- alpha mRNA increase the expression of.Ala-Gln can upregulate SLC15A1mRNA BIECs after culture found phosphatidylinositol 3 kinase (phosphatidylinositol, 3-kinase, PI3K) / protein kinase B (protein kinase B, AKT) expression of signal pathway key genes are significantly increased (P0.05). In summary, SLC15A1, SLC15A2, SLC15A3 and SLC15A4 are widely expressed in adult cows, but they the expression level in different organs and tissues of.SLC15A1 mRNA expression in BIECs was the highest, mainly through the Cdx2 and PPAR- alpha gene regulation.Ala-Gln upregulates the small peptide transporter The expression of the key genes in the PI3K/AKT conduction signal is up-regulated.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823

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