N-乙酰基-D-半乳糖胺缓解大豆凝集素对猪小肠上皮屏障损伤的研究

发布时间：2018-03-22 15:51

本文选题：大豆凝集素　切入点：N-乙酰基-D-半乳糖胺　出处：《吉林农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：影响大豆品质的主要抗营养因子之一即为大豆凝集素,研究大豆凝集素的拮抗物,对降低大豆抗营养作用和提高利用效率具有重要意义。本文通过两个试验,研究拮抗物N-乙酰基-D-半乳糖胺能否有效减弱大豆凝集素对仔猪小肠上皮细胞机械屏障的损伤。试验一:N-乙酰基-D-半乳糖胺减弱大豆凝集素对猪小肠上皮细胞通透性的影响。本试验预先加入浓度为0,0.125×10-4,0.25×10-4,0.5×10-4,1.0×10-4,2.0×10-4mmol/l的N-乙酰基-D-半乳糖胺,处理细胞1h、2h、4h、8h后,再加入0.5mg/ml的大豆凝集素处理24h。测定跨上皮细胞电阻值(TEER)和细胞培养液中碱性磷酸酶(AP)的活性,来反映细胞膜通透性和完整性的变化情况。试验结果表明:N-乙酰基-D-半乳糖胺减缓了细胞TEER值下降,也降低了细胞培养液中AP的活性。试验二:N-乙酰基-D-半乳糖胺减弱大豆凝集素对猪小肠上皮细胞紧密连接蛋白及其基因相对表达量的影响。紧密连接蛋白相对表达量部分,本试验采用蛋白免疫印迹法,对紧密连接蛋白Occludin和Claudin-3相对表达量变化进行检测。紧密连接相关基因相对表达量部分,本试验采用Real Time Quantitative PCR中的SYBR Green I荧光染料嵌合法,对紧密连接Occludin和Claudin-3 mRNA相对表达量变化进行检测。试验通过空白组(不添加任何刺激物)、对照组(只加大豆凝集素)和处理组(先加拮抗物再加大豆凝集素)三组比较,紧密连接蛋白相对表达量和其相关基因相对表达量的变化情况。试验结果表明:对照组细胞间紧密连接蛋白Occludin和Claudin-3的相对表达量分别降低了17.8%和43.5%(P0.05)。处理组细胞间紧密连接蛋白Occludin和Claudin-3的相对表达量分别降低了4.3%和7.2%(P0.05)。对照组Occludin mRNA和Claudin-3 mRNA表达量分别下降了17.3%(P0.01)和42%(P0.01)。处理组Occludin mRNA和Claudin-3 mRNA表达量分别下降了1.6%(P0.05)和2.7%(P0.01)。综上所述:在体外培养条件下,适宜浓度的N-乙酰基-D-半乳糖胺抑制了大豆凝集素对仔猪小肠上皮细胞屏障通透性的影响。大豆凝集素抑制了小肠上皮细胞紧密连接蛋白的合成,加入拮抗物N-乙酰基-D-半乳糖胺后减弱了大豆凝集素对细胞紧密连接蛋白合成的抑制作用。适宜浓度的N-乙酰基-D-半乳糖胺能够减弱大豆凝集素对仔猪小肠上皮细胞屏障功能的损伤作用。
[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.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S816

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