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乳腺中葡萄糖摄取主要依赖葡萄糖转运蛋白(glucose transporter,GLUT),不同GLUT对葡萄糖摄取所起作用不同。为研究GLUT1、GLUT3和GLUT8参与牦牛(Bos grunniens)泌乳机能调节的分子机制,本研究采用胰酶消化法离体培养牦牛乳腺上皮细胞,通过免疫细胞化学方法检测角蛋白18、β-酪蛋白和波形蛋白;从纯化的细胞中分别克隆牦牛GLUT1、GLUT3和GLUT8基因,并分析其相关生物学特性;采用q RT-PCR和Western blot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。结果显示,角蛋白18和β-酪蛋白在分离得到的细胞中表达,波形蛋白不表达,所分离的细胞为乳腺上皮细胞。成功克隆出包含完整编码区的牦牛GLUT1、GLUT3和GLUT8基因序列(Gen Bank登录号分别为:KU902419,KX094556和KX268646),其编码区核苷酸长度分别为1479、1 485和1 437 bp,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。在乳腺上皮细胞中,GLUT1的表达量最高,GLUT8的表达量次之,GLUT3的表达量最低,且三者之间的表达差异极显著(P<0.01)。牦牛乳腺上皮细胞胞质和胞核均有GLUT1、GLUT3和GLUT8蛋白表达,且主要分布在细胞核。研究结果为了解牦牛GLUT生理学功能提供了重要信息,为进一步研究其调控牦牛泌乳机能的生物学作用提供了新的理论依据。
Glucose uptake in the mammary gland depends mainly on the glucose transporter (GLUT), and different GLUTs have different effects on glucose uptake. In order to investigate the molecular mechanism of GLUT1, GLUT3 and GLUT8 involved in the regulation of lactation in Bos grunniens, yak mammary epithelial cells were cultured in vitro by trypsin digestion and detected by immunocytochemistry for the detection of keratin 18, β-casein and The expression of GLUT1, GLUT3 and GLUT8 genes in yaks were analyzed respectively from the purified cells. The relative biological characteristics of yak GLUT1, GLUT3 and GLUT8 were analyzed by q RT-PCR and Western blot respectively. , Indirect immunofluorescence assay in breast epithelial cells in the distribution. The results showed that keratin 18 and β-casein were expressed in isolated cells, while vimentin was not expressed. The isolated cells were mammary epithelial cells. The sequences of the GLUT1, GLUT3 and GLUT8 genes of yak including the complete coding region were successfully cloned (GenBank accession numbers: KU902419, KX094556 and KX268646, respectively). The nucleotide lengths of the yak GLUT1, GLUT3 and GLUT8 genes were 1479, 1 485 and 1 437 bp, Encoding 492, 494 and 478 amino acids. All of them were highly conserved during evolution. The encoded proteins had similar physicochemical properties and all had 12 membrane-spanning hydrophobic membrane proteins. In breast epithelial cells, the highest expression of GLUT1, followed by the expression of GLUT8, GLUT3 the lowest expression, and the difference between the three was extremely significant (P <0.01). The yak mammary gland epithelial cells have cytoplasmic and nuclear GLUT1, GLUT3 and GLUT8 protein expression, and 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 its biological role in regulating yak lactation function.