【摘要】：為了研究葡萄糖轉(zhuǎn)運蛋白1(Glucose transporter1,GLUT1)、葡萄糖轉(zhuǎn)運蛋白3(GLUT3)和葡萄糖轉(zhuǎn)運蛋白8(GLUT8)參與牦牛泌乳機(jī)能調(diào)節(jié)的分子機(jī)制,以及EGF(表皮生長因子,Epidermal growth factor)和INS(胰島素,Insulin)對葡萄糖代謝的調(diào)控。本研究首先采用改良的聯(lián)合酶消化組織塊法體外培養(yǎng)了牦牛乳腺上皮細(xì)胞,從純化的細(xì)胞中分別克隆牦牛GLUT1、GLUT3和GLUT8基因并分析其生物學(xué)特性;采用RT-qPCR和Western-blotting分別從基因和蛋白水平分析三者在乳腺上皮細(xì)胞表達(dá)差異性,間接免疫熒光檢測其在乳腺上皮細(xì)胞中的分布;用RT-qPCR和Western-blotting方法檢測了EGF和INS對GLUT1基因和蛋白表達(dá)的影響。取得了如下成果:(1)體外成功培養(yǎng)了牦牛乳腺上皮細(xì)胞,并確定了適合其冷凍保存的方法。(2)首次克隆出了牦牛GLUT1、GLUT3和GLUT8基因(GenBank登錄號分別為:KU902419,KX094556和KX268646)。它們分別編碼492、494和478個氨基酸,三者在進(jìn)化過程中均有較高的保守性,編碼的蛋白具有相似理化性質(zhì),均具有12個跨膜螺旋區(qū)的疏水性膜蛋白。(3)GLUT1、GLUT3和GLUT8在乳腺上皮細(xì)胞均有表達(dá),GLUT1的表達(dá)量最高,GLUT8的表達(dá)量次之,GLUT3的表達(dá)量最低,且三者之間表達(dá)差異極顯著(P0.01);另外,三者在牦牛乳腺上皮細(xì)胞胞質(zhì)和胞核均有表達(dá),且主要分布在細(xì)胞核。(4)在牦牛乳腺上皮細(xì)胞培養(yǎng)液中加入不同濃度的EGF和INS,當(dāng)EGF濃度為50 ng/mL時,GLUT1的相對表達(dá)量最高,且與其它各組之間差異顯著(P0.05);隨著EGF濃度的升高,GLUT1的相對表達(dá)量降低,且都低于對照組(EGF濃度為0 ng/m L)。表明當(dāng)外源因子超過一定的濃度范圍時,會抑制相關(guān)基因及蛋白的表達(dá),同時也說明EGF促進(jìn)GLUT1表達(dá)的最佳濃度為50 ng/m L。當(dāng)INS濃度為500ng/m L時,GLUT1的相對表達(dá)量最高,且與其它各組之間差異顯著(P0.05),提示INS對牦牛乳腺上皮細(xì)胞GLUT1的表達(dá)具有促進(jìn)作用,且具有濃度依賴性。本研究發(fā)現(xiàn)EGF和INS均可在體外調(diào)控牦牛乳腺上皮細(xì)胞中GLUT1基因和蛋白的表達(dá),提示其在一定程度上能通過調(diào)控GLUT1的表達(dá)參與葡萄糖代謝調(diào)節(jié),為進(jìn)一步研究GLUT1調(diào)控牦牛泌乳功能的生物學(xué)作用提供了新的理論依據(jù)。
[Abstract]:To study the molecular mechanism of glucose transporter 1 (Glucose transporter1,GLUT1), glucose transporter 3 (GLUT3) and glucose transporter 8 (GLUT8) involved in the regulation of lactation function in yaks, and the regulation of glucose metabolism by EGF (epidermal growth factor) and INS). In this study, yak mammary gland epithelial cells were cultured in vitro by a modified enzyme digestion method. The yak GLUT1,GLUT3 and GLUT8 genes were cloned from the purified cells and their biological characteristics were analyzed. RT-qPCR and Western-blotting were used to analyze the difference of expression of the three proteins in mammary epithelial cells from the level of gene and protein, and indirect immunofluorescence was used to detect their distribution in mammary epithelial cells. The effects of EGF and INS on the expression of GLUT1 gene and protein were detected by RT-qPCR and Western-blotting. The results are as follows: (1) Yak mammary epithelial cells were successfully cultured in vitro, and the suitable cryopreservation methods were determined. (2) the yak GLUT1,GLUT3 and GLUT8 genes were cloned for the first time (GenBank accession numbers were: KU902419 KX094556 and KX268646). They encode 492494 amino acids and 492494 amino acids respectively. The three amino acids are highly conserved in the evolution process, and the encoded proteins have similar physicochemical properties. Hydrophobic membrane proteins with 12 transmembrane helical regions were found. (3) GLUT1,GLUT3 and GLUT8 had the highest level of GLUT1 expression in breast epithelial cells, followed by the lowest level of GLUT8 expression, and the difference between them was significant (P0.01). The three proteins were expressed in cytoplasm and nucleus of mammary epithelial cells of yak, and mainly distributed in the nucleus. (4) the relative expression of GLUT1 was the highest when the concentration of EGF was 50 ng/mL by adding different concentrations of EGF and INS, into the culture medium of yak mammary epithelial cells. The relative expression of GLUT1 decreased with the increase of EGF concentration and was lower than that of the control group (EGF concentration was 0 ng/m L). The results showed that when the exogenous factors exceeded a certain concentration range, the expression of related genes and proteins was inhibited, and the optimal concentration of EGF to promote the expression of GLUT1 was 50 ng/m / L. When the concentration of INS was 500ng/m L, the relative expression of GLUT1 was the highest, and the difference was significant (P0.05), which suggested that INS could promote the expression of GLUT1 in Yak mammary epithelial cells in a concentration-dependent manner. It was found that both EGF and INS could regulate the expression of GLUT1 gene and protein in the mammary epithelial cells of yak in vitro, suggesting that both of them could participate in the regulation of glucose metabolism by regulating the expression of GLUT1. It provides a new theoretical basis for the further study of the biological function of GLUT1 in regulating the lactation function of yaks.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S823

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