Regulation Of USF1 On The Expression Of GLUT5 And SGLT1 In Intestinal Epithelial Cells Of Chicken

In the study, we found some binding sites of transcription factor USF1 on the 1500bp of gene GLUT5 and SGLT1 5’upstream by means of ModuleMasterl.4 and TF bind. In order to have a further study on the regulatory effect of USF1 on monosaccharide transporters gene GLUTS and SGLTl, we chose primary Intestinal Epithelial Cells of Chicken as a study model in vitro. We optimized the culture conditions in vitro of chicken intestinal epithelial cells, which could provide an ideal experimental model for the study of chicken intestinal digestion and absorption mechanism. Besides, recombinant vector pcDNA3.1-USFl was used to transfect intestinal epithelial cells, which resulted over-expression of USF1 and the absolute expression of gene GLUTS and SGLT1 were tested by Real-time PCR in order to find the changes of their expression. The study could provide an important theoretical basis for chicken intestinal nutrient molecular regulation mechanism.(1) The results of optimized conditions of chicken intestinal epithelial cells primary culture revealed that:① Selection of digestive enzyme:after digested by trypsin, the IEC we got were mostly single. The adherence cells were very little, and over time, the cells died gradually; after digested by collagenase I, the vast majority of IEC we got were single, and there were minority cell rolls. The adherence cells were more, and the number of TEC was increased slowly in the period of 1 d to 3 d. But, the cells began die gradually after it was cultured 3 d; after digested by thermolysin, the vitality of EEC was great. After cultured 1 d, IEC were distributed as the shape of islands. The number of IEC was increased obviously in the period of 3 d to 7 d. The IEC could grow stably about 12 d. ② Addition level of glucose in DMEM:at the 4 levels which were 5.6、10、20 and 25 mmol/L of sugar concentration, IEC were distributed as the shape of islands after cultured 1 d, and over time, the influence of sugar concentration on the state of EEC was more and more obvious. The number of adherence cells when the sugar concentration was 5.6、10 mmol/L was more than other two high-glucose groups. Besides, the refractivity of two low-glucose groups cells greater than other two high-glucose groups. The numbers of IEC of two low-glucose groups were more than others, and IEC were distributed as the alveolate shapes.(2) The Real-time PCR results showed that: ① The expression of gene SGLT1 of transfected group was significantly higher than negative control group and blank control group (P<0.01); ② The expression of gene GLUT5 in three groups was not significant (P>0.05).In conclusion:(1) Thermolysin was the best-fit enzyme to digest the intestinal of chicken. (2) The best-fit glucose concentration of IEC was 5.6 mmol/L. (3) The over-expression of Gene USF1 played a positive regulation on the expression of Monosaccharide transporter gene SGLT1.