EGF和胰岛素对牦牛乳腺上皮细胞主要葡萄糖运载体表达的影响
发布时间：2018-09-19 18:46
【摘要】：为了研究葡萄糖转运蛋白1(Glucose transporter1,GLUT1)、葡萄糖转运蛋白3(GLUT3)和葡萄糖转运蛋白8(GLUT8)参与牦牛泌乳机能调节的分子机制,以及EGF(表皮生长因子,Epidermal growth factor)和INS(胰岛素,Insulin)对葡萄糖代谢的调控。本研究首先采用改良的联合酶消化组织块法体外培养了牦牛乳腺上皮细胞,从纯化的细胞中分别克隆牦牛GLUT1、GLUT3和GLUT8基因并分析其生物学特性;采用RT-qPCR和Western-blotting分别从基因和蛋白水平分析三者在乳腺上皮细胞表达差异性,间接免疫荧光检测其在乳腺上皮细胞中的分布;用RT-qPCR和Western-blotting方法检测了EGF和INS对GLUT1基因和蛋白表达的影响。取得了如下成果:(1)体外成功培养了牦牛乳腺上皮细胞,并确定了适合其冷冻保存的方法。(2)首次克隆出了牦牛GLUT1、GLUT3和GLUT8基因(GenBank登录号分别为:KU902419,KX094556和KX268646)。它们分别编码492、494和478个氨基酸,三者在进化过程中均有较高的保守性,编码的蛋白具有相似理化性质,均具有12个跨膜螺旋区的疏水性膜蛋白。(3)GLUT1、GLUT3和GLUT8在乳腺上皮细胞均有表达,GLUT1的表达量最高,GLUT8的表达量次之,GLUT3的表达量最低,且三者之间表达差异极显著(P0.01);另外,三者在牦牛乳腺上皮细胞胞质和胞核均有表达,且主要分布在细胞核。(4)在牦牛乳腺上皮细胞培养液中加入不同浓度的EGF和INS,当EGF浓度为50 ng/mL时,GLUT1的相对表达量最高,且与其它各组之间差异显著(P0.05);随着EGF浓度的升高,GLUT1的相对表达量降低,且都低于对照组(EGF浓度为0 ng/m L)。表明当外源因子超过一定的浓度范围时,会抑制相关基因及蛋白的表达,同时也说明EGF促进GLUT1表达的最佳浓度为50 ng/m L。当INS浓度为500ng/m L时,GLUT1的相对表达量最高,且与其它各组之间差异显著(P0.05),提示INS对牦牛乳腺上皮细胞GLUT1的表达具有促进作用,且具有浓度依赖性。本研究发现EGF和INS均可在体外调控牦牛乳腺上皮细胞中GLUT1基因和蛋白的表达,提示其在一定程度上能通过调控GLUT1的表达参与葡萄糖代谢调节,为进一步研究GLUT1调控牦牛泌乳功能的生物学作用提供了新的理论依据。
[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.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823
[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.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823
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