本文選題：成年奶牛 + SLC15家族��； 參考：《揚州大學》2017年碩士論文

【摘要】：質(zhì)子轉(zhuǎn)運載體15家族(Solute carrier 15 family,SLC15)又被稱為小肽轉(zhuǎn)運載體家族,主要位于細胞膜上,能夠利用電化學梯度將短鏈肽和擬肽類物質(zhì)轉(zhuǎn)運進入細胞內(nèi)。SLC15家族成員主要包括SLC15A1、SLC15A2、SLC15A3和SLC15A4,目前鮮有報道能夠全面地研究這些小肽轉(zhuǎn)運載體在成年奶牛上的表達差異。小肽轉(zhuǎn)運載體能夠轉(zhuǎn)運腸道中的營養(yǎng)物質(zhì),它也受這些營養(yǎng)物質(zhì)調(diào)控。丙氨酰谷氨酰胺二肽(alanyl-glutamine,Ala-Gln)作為小腸的主要能源物質(zhì),它對腸道中小肽轉(zhuǎn)運載體的調(diào)控也鮮有研究。因此本試驗研究SLC15A1、SLC15A2、SLC15A3和SLC15A4在成年奶牛組織器官中的表達情況以及Ala-Gln對奶牛小腸上皮細胞(Bovine intestinal epithelial cells,BIECs)中小肽轉(zhuǎn)運載體的調(diào)控作用,為SLC15家族在成年奶牛中的生物學功能以及Ala-Gln對BIECs的調(diào)控機制提供科學依據(jù)。試驗一、小肽轉(zhuǎn)運載體在成年奶牛組織器官中的表達試驗選取3頭健康的成年荷斯坦奶牛進行屠宰,采集心臟、肝臟、脾臟、肺臟、腎臟、瘤胃、網(wǎng)胃、瓣胃、皺胃、十二指腸、空腸、回腸、盲腸、結(jié)腸和直腸。試驗采用實時熒光定量PCR法對器官組織中的SLC15A1、SLC15A2、SLC15A3和SLC15A4 mRNA進行分析;采用Western Blot方法對器官組織中的SLC15A1、SLC15A2和SLC15A4蛋白進行分析。試驗結(jié)果發(fā)現(xiàn)SLC15A1、SLC15A2、SLC15A3和SLC15A4mRNA在器官組織中均能表達,但是在回腸中沒有發(fā)現(xiàn)SLC15A2 mRNA的表達。SLC15A1在成年奶牛的肺臟、腎臟和空腸的表達極顯著高于其它器官組織(P0.01);SLC15A2在腎臟組織中的表達量均極顯著高于其它器官組織(P0.01);SLC15A3mRNA在瘤胃、脾臟、結(jié)腸和肺臟中的表達極顯著高于其它器官組織(P0.01);SLC15A4在脾臟,腎臟,肝臟,盲腸,結(jié)腸和肺臟中均有較高的表達水平。試驗二、奶牛小腸上皮細胞原代培養(yǎng)及其小肽轉(zhuǎn)運載體的表達在無菌條件下分離奶牛小腸上皮組織,用胰酶消化獲得BIECs并純化,免疫熒光和瓊脂糖凝膠電泳鑒定BIECs,實時熒光定量PCR檢測細胞內(nèi)SLC15A1、SLC15A2、SLC15A3和 SLC15A4 mRNA 表達;Western Blot 檢測細胞中 SLC15A1、SLC15A2 和 SLC15A4 蛋白表達。結(jié)果表明,BIECs的細胞質(zhì)中均含有細胞角蛋白18熒光,在細胞中檢測到絨毛蛋白、上皮細胞鈣粘蛋白和小腸脂肪酸結(jié)合蛋白基因的表達證實細胞為純化的BIECs。細胞中SLC15A1 mRNA 的表達量極顯著高于 SLC15A2、SLC15A3 和 SLC15A4 mRNA(P0.01)。SLC15A1蛋白的表達量最高,極顯著高于SLC15A2和SLC15A4(P0.01)。試驗三、Ala-Gln對奶牛小腸上皮細胞中小肽轉(zhuǎn)運載體表達的影響不同時間和濃度的Ala-Gln培養(yǎng)BIECs,確定最佳培養(yǎng)時間和濃度。用實時熒光定量PCR 檢測 Ala-Gln、丙氨酸(L-alanine,Ala)和谷氨酰胺(L-glutamine,Gln)對 BIECs中小肽轉(zhuǎn)運載體及其相關(guān)基因mRNA的影響,Western Blot檢測Ala-Gln、Ala和Gln對BIECs中SLC15A1、SLC15A2和SLC15A4蛋白的影響。結(jié)果發(fā)現(xiàn),Ala-Gln能夠促進BIECs增殖,其中1mMAla-Gln在4h時極顯著促進細胞增殖(P0.01),并且該組中SLC15A1 mRNA、SLC15A2 mRNA、SLC15A3 mRNA 和 SLC15A4 mRNA 的表達量極顯著高于其它各組(P0.01),顯著促進SLC15A1蛋白的表達(P0.05)。Cdx2和PPAR-α mRNA的提高能夠上調(diào)SLC15A1mRNA的表達量。Ala-Gln培養(yǎng)BIECs后發(fā)現(xiàn)磷脂酰肌醇3激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶 B(protein kinase B,AKT)信號通路關(guān)鍵基因的表達都顯著升高(P0.05)。綜上,SLC15A1、SLC15A2、SLC15A3和SLC15A4在成年奶牛中廣泛表達,但是它們在器官組織中的表達水平存在較大差異。SLC15A1 mRNA在BIECs中表達量最高,主要是通過Cdx2和PPAR-α基因調(diào)控的。Ala-Gln能夠上調(diào)小肽轉(zhuǎn)運載體的表達,并且能上調(diào)PI3K/AKT傳導信號中關(guān)鍵基因的表達。
[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.

【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S823

【參考文獻】

相關(guān)期刊論文 前10條

1 張繼杰;周安國;王之盛;;谷氨酰胺對豬腸道形態(tài)和功能的影響及其機制[J];畜禽業(yè);2007年11期

2 匡偉;王海霞;邵桃玉;趙國琦;;新生山羊小腸上皮細胞株的建立和鑒定[J];中國畜牧雜志;2007年19期

3 鄧敦;李鐵軍;黃瑞林;印遇龍;范明哲;伍國耀;鐘華宜;;小肽轉(zhuǎn)運蛋白(PepT1)及其活性調(diào)控[J];廣西農(nóng)業(yè)生物科學;2005年04期

4 李燕;林雪彥;姜運良;蘇鵬程;王中華;;應(yīng)用RT-PCR方法檢測牛消化道各段Ⅰ型肽載體mRNA差異表達的研究[J];動物營養(yǎng)學報;2008年02期

5 徐奇友;王常安;許紅;尹家勝;馬建章;;丙氨酰-谷氨酰胺對哲羅魚仔魚生長和抗氧化能力的影響[J];動物營養(yǎng)學報;2009年06期

6 李慧;格日樂其木格;王健宇;王娟;王芳;薛冰華;劉忠華;;腸絨毛消化法所得仔豬小腸上皮細胞的培養(yǎng)與鑒定[J];中國細胞生物學學報;2011年10期

7 王靜;張彥明;仝鋼;劉芳寧;周宏超;何雷;楊小云;徐彥召;洪海霞;;新生仔豬小腸上皮細胞的分離培養(yǎng)和鑒定[J];畜牧獸醫(yī)學報;2010年01期

8 周傳麗;劉錚鑄;俞英;張勤;;仔豬小腸黏膜上皮細胞體外分離培養(yǎng)及鑒定[J];中國農(nóng)業(yè)科學;2011年21期

9 陳靜;劉顯軍;張飛;邊連全;;谷氨酰胺對免疫應(yīng)激仔豬免疫器官指數(shù)的影響[J];中國獸醫(yī)雜志;2010年09期

10 王玲;劉輝;李勝利;趙勝軍;王立斌;楊再俊;;十二指腸灌注大豆小肽和氨基酸對奶山羊小腸肽吸收的影響[J];動物營養(yǎng)學報;2010年02期

相關(guān)碩士學位論文 前6條

1 郭靜;奶牛POTs的表達及小肽對乳腺上皮細胞乳蛋白合成的影響[D];揚州大學;2015年

2 劉志浩;JBP485經(jīng)PEPT1的攝取，轉(zhuǎn)運和調(diào)節(jié)[D];大連醫(yī)科大學;2011年

3 王輝;谷氨酰胺二肽在斷奶仔豬離體空腸的轉(zhuǎn)運特點和對空腸發(fā)育影響的研究[D];四川農(nóng)業(yè)大學;2010年

4 劉桂梅;牛消化道Ⅰ型肽載體（bPepTI）表達定量與真核表達技術(shù)研究[D];山東農(nóng)業(yè)大學;2009年

5 匡偉;新生山羊小腸上皮細胞系的建立[D];揚州大學;2007年

6 周榮艷;谷氨酰胺及丙氨酰谷氨酰胺對早期斷奶仔豬腸上皮細胞增殖和腸道免疫的影響[D];華中農(nóng)業(yè)大學;2004年

，

本文編號：1737779