發(fā)布時(shí)間：2018-05-12 17:59

  本文選題：雞小腸上皮細(xì)胞 + GATA3��； 參考：《山西農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：糖類是動(dòng)物生長(zhǎng)必不可少的主要營(yíng)養(yǎng)物質(zhì)之一,飲食中糖類的消化吸收對(duì)動(dòng)物的正常生長(zhǎng)發(fā)育起著重要作用。糖類的消化主要在小腸中進(jìn)行,位于腸上皮細(xì)胞刷狀緣膜上的糖類營(yíng)養(yǎng)轉(zhuǎn)運(yùn)蛋白SGLT1和GLUT5負(fù)責(zé)單糖的轉(zhuǎn)運(yùn)。所以調(diào)控兩種基因的表達(dá)可以影響糖類的吸收,進(jìn)而影響動(dòng)物的生長(zhǎng)發(fā)育。轉(zhuǎn)錄因子GATA3是GATA鋅指轉(zhuǎn)錄因子家族的一員,它對(duì)許多組織細(xì)胞中基因的表達(dá)都有調(diào)控作用。本研究為探索雞腸道中轉(zhuǎn)錄因子GATA3對(duì)sGLT1和GLUT5的調(diào)控機(jī)制。首先對(duì)雞小腸上皮細(xì)胞的體外分離培養(yǎng)的適宜條件和營(yíng)養(yǎng)因子(接種密度和血清種類)進(jìn)行篩選,通過(guò)篩選出的最佳條件培養(yǎng)細(xì)胞,并對(duì)培養(yǎng)出的細(xì)胞進(jìn)行鑒定,然后利用生物信息學(xué)軟件尋找SGLT1和GLUT5基因5’上游GATA3基因的結(jié)合位點(diǎn),在此基礎(chǔ)上通過(guò)脂質(zhì)體轉(zhuǎn)染法將實(shí)驗(yàn)室已成功構(gòu)建的真核表達(dá)載體pcDNA3.1-GATA3轉(zhuǎn)染雞小腸上皮細(xì)胞,最后利用實(shí)時(shí)熒光定量PCR技術(shù)檢測(cè)轉(zhuǎn)染后雞小腸上皮細(xì)胞中基因GATA3、SGLT1和GLUT5的絕對(duì)表達(dá)量。研究結(jié)果：體外分離培養(yǎng)雞小腸上皮細(xì)胞的最佳接種密度為6.5×105/mL左右；與添加胎牛血清的培養(yǎng)液相比,添加雞血清的培養(yǎng)液更適宜雞小腸上皮細(xì)胞的體外分離培養(yǎng)；通過(guò)篩選得到的最佳適宜條件和營(yíng)養(yǎng)因子成功體外分離培養(yǎng)出雞小腸上皮細(xì)胞,并通過(guò)鑒定得到肯定；生物信息學(xué)軟件發(fā)現(xiàn)糖類營(yíng)養(yǎng)轉(zhuǎn)運(yùn)蛋白SGLT1和GLUT5基因5’上游含有多個(gè)GATA3基因的結(jié)合位點(diǎn)；轉(zhuǎn)染組GATA3的表達(dá)量極顯著高于空白對(duì)照組和陰性對(duì)照組(P0.01),說(shuō)明利用脂質(zhì)體轉(zhuǎn)染法將真核表達(dá)載體pcDNA3.1-GATA3已成功轉(zhuǎn)染進(jìn)入雞小腸上皮細(xì)胞；雞小腸上皮細(xì)胞中GATA3過(guò)表達(dá)后,轉(zhuǎn)染組GLUT5的表達(dá)量極顯著高于空白對(duì)照和陰性對(duì)照組(P0.01),而轉(zhuǎn)染組SGLT1的表達(dá)量與對(duì)照組之間無(wú)明顯的差異(P0.05)。以上結(jié)果揭示了在原代培養(yǎng)的雞小腸上皮細(xì)胞中轉(zhuǎn)錄因子GATA3的過(guò)表達(dá)對(duì)糖類轉(zhuǎn)運(yùn)蛋白基因GLUT5的表達(dá)起到正調(diào)控作用,而對(duì)糖類營(yíng)養(yǎng)轉(zhuǎn)運(yùn)蛋白基因SGLT1則沒(méi)有影響,為研究雞腸道功能以期選育高飼料轉(zhuǎn)化率的優(yōu)良雞種提供參考依據(jù)。
[Abstract]:Carbohydrate is one of the essential nutrients for animal growth. The digestion and absorption of sugar in diet play an important role in the normal growth and development of animals. The digestion of carbohydrates mainly takes place in the small intestine, and SGLT1 and GLUT5, which are located on the brush-edge membrane of intestinal epithelial cells, are responsible for the transport of monosaccharide. Therefore, regulating the expression of two genes can affect the absorption of sugar, and then affect the growth and development of animals. Transcription factor GATA3 is a member of GATA zinc finger transcription factor family. It regulates gene expression in many tissue cells. The aim of this study was to investigate the regulatory mechanism of GATA3 on sGLT1 and GLUT5 in chicken intestinal tract. The optimal conditions and nutritional factors (inoculation density and serum species) for isolation and culture of chicken small intestinal epithelial cells in vitro were first selected. Then the binding sites of SGLT1 and GLUT5 gene 5'upstream GATA3 gene were found by bioinformatics software, and then the successfully constructed eukaryotic expression vector pcDNA3.1-GATA3 was transfected into chicken small intestinal epithelial cells by liposome transfection. In the end, the absolute expression of GATA3GLT1 and GLUT5 in transfected chicken small intestinal epithelial cells was detected by real-time fluorescence quantitative PCR. The results showed that the optimal inoculation density of chicken small intestinal epithelial cells was about 6.5 脳 105/mL, and the medium supplemented with fetal bovine serum was more suitable for isolation and culture of chicken small intestinal epithelial cells in vitro. Chicken small intestinal epithelial cells were successfully isolated and cultured in vitro under the optimum conditions and nutrition factors, and confirmed by identification. Bioinformatics software found that SGLT1 and GLUT5 gene 5'upstream contained multiple binding sites of GATA3 gene. The expression of GATA3 in the transfected group was significantly higher than that in the blank control group and the negative control group, indicating that the eukaryotic expression vector pcDNA3.1-GATA3 had been successfully transfected into the chicken intestinal epithelial cells by liposome transfection, and the expression of GATA3 in the chicken intestinal epithelial cells was over-expressed. The expression of GLUT5 in the transfected group was significantly higher than that in the blank control group and the negative control group, but there was no significant difference in the expression of SGLT1 between the transfected group and the control group. These results suggest that the overexpression of transcription factor GATA3 plays a positive role in the expression of carbohydrate transporter gene GLUT5 in primary cultured chicken small intestinal epithelial cells, but has no effect on the carbohydrate nutrition transporter gene SGLT1. To study the intestinal function of chicken in order to select good chicken breeds with high feed conversion rate.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S831

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本文編號(hào)：1879583