本文選題：大豆凝集素　切入點(diǎn)：N-乙酰基-D-半乳糖胺　出處：《吉林農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：影響大豆品質(zhì)的主要抗?fàn)I養(yǎng)因子之一即為大豆凝集素,研究大豆凝集素的拮抗物,對降低大豆抗?fàn)I養(yǎng)作用和提高利用效率具有重要意義。本文通過兩個試驗(yàn),研究拮抗物N-乙�；�-D-半乳糖胺能否有效減弱大豆凝集素對仔豬小腸上皮細(xì)胞機(jī)械屏障的損傷。試驗(yàn)一:N-乙�；�-D-半乳糖胺減弱大豆凝集素對豬小腸上皮細(xì)胞通透性的影響。本試驗(yàn)預(yù)先加入濃度為0,0.125×10-4,0.25×10-4,0.5×10-4,1.0×10-4,2.0×10-4mmol/l的N-乙�；�-D-半乳糖胺,處理細(xì)胞1h、2h、4h、8h后,再加入0.5mg/ml的大豆凝集素處理24h。測定跨上皮細(xì)胞電阻值(TEER)和細(xì)胞培養(yǎng)液中堿性磷酸酶(AP)的活性,來反映細(xì)胞膜通透性和完整性的變化情況。試驗(yàn)結(jié)果表明:N-乙酰基-D-半乳糖胺減緩了細(xì)胞TEER值下降,也降低了細(xì)胞培養(yǎng)液中AP的活性。試驗(yàn)二:N-乙�；�-D-半乳糖胺減弱大豆凝集素對豬小腸上皮細(xì)胞緊密連接蛋白及其基因相對表達(dá)量的影響。緊密連接蛋白相對表達(dá)量部分,本試驗(yàn)采用蛋白免疫印跡法,對緊密連接蛋白Occludin和Claudin-3相對表達(dá)量變化進(jìn)行檢測。緊密連接相關(guān)基因相對表達(dá)量部分,本試驗(yàn)采用Real Time Quantitative PCR中的SYBR Green I熒光染料嵌合法,對緊密連接Occludin和Claudin-3 mRNA相對表達(dá)量變化進(jìn)行檢測。試驗(yàn)通過空白組(不添加任何刺激物)、對照組(只加大豆凝集素)和處理組(先加拮抗物再加大豆凝集素)三組比較,緊密連接蛋白相對表達(dá)量和其相關(guān)基因相對表達(dá)量的變化情況。試驗(yàn)結(jié)果表明:對照組細(xì)胞間緊密連接蛋白Occludin和Claudin-3的相對表達(dá)量分別降低了17.8%和43.5%(P0.05)。處理組細(xì)胞間緊密連接蛋白Occludin和Claudin-3的相對表達(dá)量分別降低了4.3%和7.2%(P0.05)。對照組Occludin mRNA和Claudin-3 mRNA表達(dá)量分別下降了17.3%(P0.01)和42%(P0.01)。處理組Occludin mRNA和Claudin-3 mRNA表達(dá)量分別下降了1.6%(P0.05)和2.7%(P0.01)。綜上所述:在體外培養(yǎng)條件下,適宜濃度的N-乙�；�-D-半乳糖胺抑制了大豆凝集素對仔豬小腸上皮細(xì)胞屏障通透性的影響。大豆凝集素抑制了小腸上皮細(xì)胞緊密連接蛋白的合成,加入拮抗物N-乙�；�-D-半乳糖胺后減弱了大豆凝集素對細(xì)胞緊密連接蛋白合成的抑制作用。適宜濃度的N-乙酰基-D-半乳糖胺能夠減弱大豆凝集素對仔豬小腸上皮細(xì)胞屏障功能的損傷作用。
[Abstract]:One of the main anti-nutritional factors affecting soybean quality is soybean agglutinin. It is important to study the antagonist of soybean agglutinin to reduce the anti-nutritional effect of soybean and improve the utilization efficiency. Two experiments were carried out in this paper. To study whether the antagonist N- acetyl-Dgalactosamine can effectively attenuate the damage of soybean agglutinin to the mechanical barrier of small intestinal epithelial cells in piglets. Experiment 1: N- acetyl-D- galactosamine attenuates the effect of soybean agglutinin on porcine intestinal epithelial cells. In this experiment, the concentration of 0.125 脳 10 ~ (-4) (0.25 脳 10 ~ (-4)) C _ (0.5) 脳 10 ~ (-4) C _ (0.5) 脳 10 ~ (-4) C _ (1.0 脳 10 ~ (-4)) 10-4mmol/l N acetyl-Dgalactosamine was added. The cells were treated for 1 h, 2 h and 4 h for 8 h, then treated with soybean agglutinin (0.5mg/ml) for 24 h. The activity of transepithelial cell resistance and alkaline phosphatase (AP) in the cell culture medium was measured. To reflect the changes in cell membrane permeability and integrity. The results showed that the cell TEER value was slowed down by 1: N-acetyl-Dgalactosine. The effects of soybean lectin on the relative expression of tight junction protein and its gene in porcine small intestinal epithelial cells were attenuated in experiment 2: N-acetyl-Dgalactosamine. In this experiment, the relative expression of Occludin and Claudin-3 was detected by Western blot. The relative expression of Real Time Quantitative PCR was detected by SYBR Green I dye embedding method. The changes of relative expression of tightly connected Occludin and Claudin-3 mRNA were measured. The results were compared among three groups: blank group (without adding any irritant), control group (with soybean agglutinin only) and treatment group (with antagonist and then soybean agglutinin). The results showed that the relative expression of Occludin and Claudin-3 in the control group decreased by 17.8% and 43.5%, respectively. The relative expression of Occludin and Claudin-3 decreased by 4.3% and 7.2%, respectively. The expression of Occludin mRNA and Claudin-3 mRNA in the control group decreased by 17.3P 0.01 and 42% respectively. In the treatment group, Occludin mRNA and Claudin-3 mRNA decreased by 1.6% P0.05) and 2.7% P0.01, respectively. In conclusion, the expression of Occludin mRNA and Claudin-3 mRNA decreased by 1.6% and 2.7% P0.01in vitro culture, respectively. The effect of soybean agglutinin on the barrier permeability of intestinal epithelial cells in piglets was inhibited by the appropriate concentration of N-acetyl-Dgalactosamine. Soybean agglutinin inhibited the synthesis of tight junction protein in intestinal epithelial cells. The inhibitory effect of soybean agglutinin on cell tight junction protein synthesis was weakened by the addition of antagonist N- acetyl-Dgalactosamine. The suitable concentration of N- acetyl-D- galactosamine could attenuate the effect of soybean agglutinin on intestinal epithelium of piglets. Damage to cell barrier function.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S816

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 朱翠;師子彪;蔣宗勇;王麗;;乳酸桿菌在調(diào)節(jié)腸道屏障功能中的作用[J];中國畜牧獸醫(yī);2012年09期

2 崔巍;馬力;聞穎;劉沛;;暴發(fā)性肝衰竭時腸上皮細(xì)胞間緊密連接蛋白o(hù)ccludin表達(dá)下降[J];世界華人消化雜志;2006年31期

相關(guān)碩士學(xué)位論文 前1條

1 王幸;大豆凝集素的分離純化和細(xì)胞凝集活性的研究[D];南京農(nóng)業(yè)大學(xué);2006年

，

本文編號：1649329