【摘要】：本研究通過ModuleMaster1.4和TF bind等結(jié)合位點預(yù)測軟件,分別在GLUT5、 SGLT1上游5'調(diào)控區(qū)1500bp范圍內(nèi)找到多個USF1結(jié)合位點,進(jìn)一步研究轉(zhuǎn)錄因子USF1對GLUT5、SGLT1的調(diào)控作用；選擇原代雞腸上皮細(xì)胞為體外研究模型,優(yōu)化雞小腸上皮細(xì)胞體外分離培養(yǎng)條件,為雞小腸消化和吸收等作用機(jī)制的研究提供理想的試驗?zāi)Ｐ停徊⒅亟M載體pcDNA3.1-USF1轉(zhuǎn)染雞IEC,使USF1過表達(dá),通過實時熒光定量PCR檢測比較轉(zhuǎn)染后各試驗組GLUT5、SGLT1基因表達(dá)量變化,為雞腸道糖營養(yǎng)物質(zhì)的分子調(diào)控機(jī)理提供重要的理論依據(jù)。(1)優(yōu)化雞IEC原代培養(yǎng)條件。結(jié)果顯示：①消化酶的選擇,經(jīng)胰蛋白酶消化得到的IEC多為單個細(xì)胞,貼壁生長的細(xì)胞最少,并且隨時間的推移,細(xì)胞逐漸死亡；經(jīng)膠原酶Ⅰ消化液消化得到的IEC絕大部分是單個細(xì)胞,只有少數(shù)較小的細(xì)胞團(tuán),貼壁生長的細(xì)胞較多,培養(yǎng)至3d期間活細(xì)胞數(shù)也有所增加,但在3d之后,細(xì)胞開始逐漸死亡；經(jīng)嗜熱菌蛋白酶消化得到的細(xì)胞活力最大,當(dāng)培養(yǎng)1d后細(xì)胞呈“島嶼狀”分布,在7d時,IEC數(shù)量增多,并可穩(wěn)定生長至11d左右。②DMEM培養(yǎng)液中葡萄糖添加水平,5.6、’10、20和25 mmol/L4種糖濃度水平條件下,培養(yǎng)1d時,IEC均呈“島嶼狀”分布,隨著時間的推移,糖濃度對IEC狀態(tài)的影響越來越明顯；第3d時,糖濃度為5.6、10mmol/L的低糖組IEC貼壁生長數(shù)較高糖組多,且細(xì)胞折光性大于高糖組；培養(yǎng)至第7d時,低糖組的IEC數(shù)量明顯大于高糖組,細(xì)胞呈“蜂窩狀”結(jié)構(gòu)生長。(2)熒光定量PCR結(jié)果顯示：①轉(zhuǎn)錄因子USF1過表達(dá)后,質(zhì)粒轉(zhuǎn)染組SGLT1 mRNA表達(dá)量極顯著高于陰性對照組、空白對照組(P0.01)；②轉(zhuǎn)錄因子USF1過表達(dá)后,質(zhì)粒轉(zhuǎn)染組GLUT5 mRNA表達(dá)量與空白對照組、陰性對照組之間差異不顯著(P0.05)。綜上所述：(1)嗜熱菌蛋白酶是雞小腸的最適消化酶；(2)培養(yǎng)雞IEC的最佳葡萄糖濃度水平是5.6 mmol/L;(3)轉(zhuǎn)錄因子USFl過表達(dá)對糖轉(zhuǎn)運蛋白SGLT1基因起顯著正調(diào)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.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S831;Q78

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