【摘要】：乳腺中葡萄糖攝取主要依賴葡萄糖轉(zhuǎn)運蛋白(glucose transporter,GLUT),不同GLUT對葡萄糖攝取所起作用不同。為研究GLUT1、GLUT3和GLUT8參與牦牛(Bos grunniens)泌乳機能調(diào)節(jié)的分子機制,本研究采用胰酶消化法離體培養(yǎng)牦牛乳腺上皮細胞,通過免疫細胞化學(xué)方法檢測角蛋白18、β-酪蛋白和波形蛋白;從純化的細胞中分別克隆牦牛GLUT1、GLUT3和GLUT8基因,并分析其相關(guān)生物學(xué)特性;采用q RT-PCR和Western blot分別從基因和蛋白水平分析三者在乳腺上皮細胞表達差異性,間接免疫熒光檢測其在乳腺上皮細胞中的分布。結(jié)果顯示,角蛋白18和β-酪蛋白在分離得到的細胞中表達,波形蛋白不表達,所分離的細胞為乳腺上皮細胞。成功克隆出包含完整編碼區(qū)的牦牛GLUT1、GLUT3和GLUT8基因序列(Gen Bank登錄號分別為:KU902419,KX094556和KX268646),其編碼區(qū)核苷酸長度分別為1479、1 485和1 437 bp,分別編碼492、494和478個氨基酸;三者在進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。在乳腺上皮細胞中,GLUT1的表達量最高,GLUT8的表達量次之,GLUT3的表達量最低,且三者之間的表達差異極顯著(P0.01)。牦牛乳腺上皮細胞胞質(zhì)和胞核均有GLUT1、GLUT3和GLUT8蛋白表達,且主要分布在細胞核。研究結(jié)果為了解牦牛GLUT生理學(xué)功能提供了重要信息,為進一步研究其調(diào)控牦牛泌乳機能的生物學(xué)作用提供了新的理論依據(jù)。
[Abstract]:Glucose uptake in mammary glands is mainly dependent on glucose transporter glut, and different GLUT have different effects on glucose uptake. In order to study the molecular mechanism of GLUT3 and GLUT8 involved in the regulation of lactation function of yak (Bos grunniens), the mammary epithelial cells of yak were cultured by trypsin digestion in vitro. Keratin 18, 尾 -casein and vimentin were detected by immunocytochemistry. The GLUT3 and GLUT8 genes were cloned from purified cells, and their biological characteristics were analyzed. The difference of expression of GLUT3 gene and GLUT8 gene in mammary epithelial cells was analyzed by Q RT-PCR and Western blot from gene and protein levels, respectively. Indirect immunofluorescence was used to detect its distribution in mammary epithelial cells. The results showed that keratin 18 and 尾-casein were expressed in the isolated cells, but vimentin was not expressed. The cells isolated were mammary epithelial cells. The complete coding region of Yak GLUT1G UT3 and GLUT8 gene were cloned successfully. The (Gen Bank accession numbers of GLUT3 and GLUT8 gene were: 1: KU902419 KX094556 and KX268646). The nucleotide length of the coding region was 1479bp1 485 and 1 437bprespectively, encoding 492494 and 478amino acids, respectively. The three proteins are highly conserved in the evolution process and have similar physicochemical properties and 12 hydrophobic membrane proteins with 12 transmembrane helical regions. The highest expression of GLUT1 was found in mammary epithelial cells and the lowest was GLUT8, and the difference among them was significant (P0.01). GLUT1, GLUT3 and GLUT8 protein were expressed in cytoplasm and nucleus of mammary epithelial cells of yak, and were mainly distributed in the nucleus. The results provide important information for understanding the physiological function of yak GLUT, and provide a new theoretical basis for further study on the biological function of regulating the lactation function of yaks.
【作者單位】： 甘肅農(nóng)業(yè)大學(xué)動物醫(yī)學(xué)院/甘肅省牛羊胚胎工程技術(shù)研究中心;
【基金】：國家自然科學(xué)基金(No.31472244) 甘肅省生物技術(shù)專項(GNSW-2013-23)
【分類號】：S823.85

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