USF1调控鸡小肠上皮细胞中GLUT5和SGLT1表达的研究

发布时间：2018-08-19 13:47

【摘要】：本研究通过ModuleMaster1.4和TF bind等结合位点预测软件,分别在GLUT5、 SGLT1上游5'调控区1500bp范围内找到多个USF1结合位点,进一步研究转录因子USF1对GLUT5、SGLT1的调控作用；选择原代鸡肠上皮细胞为体外研究模型,优化鸡小肠上皮细胞体外分离培养条件,为鸡小肠消化和吸收等作用机制的研究提供理想的试验模型；并将重组载体pcDNA3.1-USF1转染鸡IEC,使USF1过表达,通过实时荧光定量PCR检测比较转染后各试验组GLUT5、SGLT1基因表达量变化,为鸡肠道糖营养物质的分子调控机理提供重要的理论依据。(1)优化鸡IEC原代培养条件。结果显示：①消化酶的选择,经胰蛋白酶消化得到的IEC多为单个细胞,贴壁生长的细胞最少,并且随时间的推移,细胞逐渐死亡；经胶原酶Ⅰ消化液消化得到的IEC绝大部分是单个细胞,只有少数较小的细胞团,贴壁生长的细胞较多,培养至3d期间活细胞数也有所增加,但在3d之后,细胞开始逐渐死亡；经嗜热菌蛋白酶消化得到的细胞活力最大,当培养1d后细胞呈“岛屿状”分布,在7d时,IEC数量增多,并可稳定生长至11d左右。②DMEM培养液中葡萄糖添加水平,5.6、’10、20和25 mmol/L4种糖浓度水平条件下,培养1d时,IEC均呈“岛屿状”分布,随着时间的推移,糖浓度对IEC状态的影响越来越明显；第3d时,糖浓度为5.6、10mmol/L的低糖组IEC贴壁生长数较高糖组多,且细胞折光性大于高糖组；培养至第7d时,低糖组的IEC数量明显大于高糖组,细胞呈“蜂窝状”结构生长。(2)荧光定量PCR结果显示：①转录因子USF1过表达后,质粒转染组SGLT1 mRNA表达量极显著高于阴性对照组、空白对照组(P0.01)；②转录因子USF1过表达后,质粒转染组GLUT5 mRNA表达量与空白对照组、阴性对照组之间差异不显著(P0.05)。综上所述：(1)嗜热菌蛋白酶是鸡小肠的最适消化酶；(2)培养鸡IEC的最佳葡萄糖浓度水平是5.6 mmol/L;(3)转录因子USFl过表达对糖转运蛋白SGLT1基因起显著正调控作用。
[Abstract]:In this study, ModuleMaster1.4 and TF bind were used to predict the binding sites, and several USF1 binding sites were found in the 1500bp region of GLUT5 and SGLT1 upstream, respectively, in order to further study the regulatory effect of USF1 on GLUT5 and SGLT1. The primary chicken intestinal epithelial cells were selected as the study model in vitro to optimize the isolation and culture conditions of chicken intestinal epithelial cells in vitro and to provide an ideal experimental model for the study of the mechanism of digestion and absorption of chicken small intestine. The recombinant vector pcDNA3.1-USF1 was transfected into chicken IECs to make USF1 overexpression. The changes of GLUT5SgLT1 gene expression in each experimental group were compared by real-time fluorescence quantitative PCR. It provides an important theoretical basis for the molecular regulation mechanism of chicken intestinal sugar nutrition. (1) optimize the primary culture conditions of chicken IEC. The results showed that the IEC digested by trypsin was a single cell, and the cells that adherent to the wall were the least, and the cells died gradually with the passage of time. The IEC digested with collagenase I was mostly a single cell, with only a few small cell clusters, more cells adherent to the wall, and the number of living cells increased during the 3 days of culture, but after 3 days, the cells began to die gradually. The activity of cells digested by thermophilic protease was the highest. After 1 day of culture, the cells were "island" distributed, and the number of IEC increased at 7 days. Under the conditions of glucose concentration of 5.6DMEM 1020 and 25 mmol/L4, the distribution of IECs was "island" when cultured for 1 day, and the distribution of IECs was "island" with the passage of time, when glucose was added to the medium for 11 days or so, and the concentration of glucose in the medium was increased steadily to the level of 5.6DMEM and 25 mmol/L4 respectively. The effect of glucose concentration on IEC state was more and more obvious. At the 3rd day, the number of IEC adherent growth in low sugar group was more than that in high glucose group, and the cell refraction was higher in low sugar group than in high glucose group, and the number of IEC in low glucose group was significantly higher than that in high glucose group at the 7th day. (2) the results of fluorescence quantitative PCR showed that the expression of SGLT1 mRNA in the plasmid transfected group was significantly higher than that in the negative control group, and that in the blank control group (P0.01), the USF1 expression of the transcription factor 2 was significantly higher than that of the negative control group. (2) the results of fluorescence quantitative PCR showed that the SGLT1 mRNA expression in the plasmid transfected group was significantly higher than that in the negative control group. The expression of GLUT5 mRNA in plasmid transfection group was not significantly different from that in blank control group and negative control group (P0.05). To sum up: (1) thermophilic protease is the optimal digestive enzyme in chicken small intestine; (2) the optimal glucose concentration of chicken IEC is 5.6 mmol / L; (3) the overexpression of transcription factor USFl plays a significant positive role in regulating the glucose transporter SGLT1 gene.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S831;Q78

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