蛋氨酸寡肽对奶牛乳腺上皮细胞酪蛋白合成相关基因表达的影响

发布时间：2018-02-10 12:17

本文关键词： 奶牛乳腺上皮细胞酪蛋白蛋氨酸寡肽基因表达　出处：《内蒙古农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：本论文以体外培养的奶牛乳腺上皮细胞(BMECs)为模型,研究不同蛋氨酸寡肽(Op-Met)浓度及Op-Met代替不同比例的游离蛋氨酸(F-Met)后对BMECs中酪蛋白基因及其相关基因表达以及细胞内外氨肽酶(AP)含量的影响,为揭示寡肽对BMECs中酪蛋白合的影响及其机理奠定理论基础。论文由三大部分构成:第一部分包含两个试验,试验一研究了不同培养时间的条件下,培养基中添加不同浓度的蛋氨酸二肽(Met-Met)和蛋氨酸三肽(Met-Met-Met)对BMECs中酪蛋白基因表达的影响,以期筛选出最适培养时间及培养浓度。利用消化法对BMECs进行原代培养,利用第3代细胞进行指标测定。将细胞随机分为11个处理组,每组5个重复,一组为对照组,试验组分别向培养基中加入终浓度为40、50、60、70和80μg/mL的Op-Met。将细胞培养24h、48h和72h。结果表明:用添加不同浓度的Op-Met的培养基培养细胞24h后,BMECs整体活力最高;当Op-Met的添加浓度为60μg/mL,培养时间为24h时,αs1-酪蛋白基因(CSN1S1)和β-酪蛋白基因(CSN2)表达量显著提高(P0.05)。试验二在确定的适宜培养时间及培养浓度后,测定细胞中两种小肽转运载体(PepTs)基因和氨肽酶氮基因(APN)的表达和细胞内外AP含量的变化。将细胞随机分为3个处理组,每组3个重复,其中一组为对照组,试验组分别加入60μg/mL的Met-Met或Met-Met-Met后将细胞培养24h。结果表明:Met-Met组的小肽转运载体2基因(PepT2)和APN表达显著提高(P0.05),细胞外的AP含量极显著升高(P0.01);Met-Met-Met组的小肽转运载体1基因(PepT1)和PepT2表达量显著提高(P0.05)。第二部分根据以前学者确定的BMECs培养基中适宜F-Met添加浓度为60μg/mL为基础,测定在培养BMECs的过程中添加适宜浓度的F-Met、Met-Met和Met-Met-Met的情况下各酪蛋白基因的表达量。将细胞随机分为4个处理组,每组3个重复,一组为对照组,试验组分别加入适宜浓度的F-Met、Met-Met或Met-Met-Met后培养细胞24h。结果表明:添加F-Met组对CSN1S1、CSN2和κ-酪蛋白基因(CSN3)均有极显著促进作用(P0.01);添加Met-Met组对CSN1S1表达有极显著的促进作用(P0.01);添加Met-Met-Met组对CSN3有极显著的抑制作用(P0.01)。第三部分测定Op-Met在替代不同比例的F-Met时,BMECs中酪蛋白基因及其相关基因表达和AP含量的变化。将细胞随机分为10个处理组,每组3个重复,一组为对照组,试验组培养基中分别添加0%、5%、10%、15%和20%的Op-Met代替其中的F-Met,培养24h。结果表明:Op-Met代替F-Met的最适代替比例为15%;当Op-Met代替量为0%时,细胞外AP含量显著升高(P0.05)。这些试验结果证明小肽的添加能使BMECs酪蛋白基因表达量提高,适宜的Op-Met添加浓度为60μg/mL时,CSN1S1和CSN2显著提高,PepT2基因表达显著提高;用Op-Met代替15%F-Met时,细胞中各酪蛋白基因表达量显著提高。
[Abstract]:In this study, the bovine mammary epithelial cells (BMECs) were cultured in vitro. The effects of different concentrations of methionine oligoseptide Op-Meta and Op-Met on the expression of casein gene and its related genes in BMECs and the content of aminopeptidase (APP) in and out of BMECs were studied. In order to reveal the effect of oligoseptide on casein synthesis in BMECs and its mechanism, the thesis consists of three parts: the first part consists of two experiments. Effects of different concentrations of methionine dipeptide Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met@@. The cells were randomly divided into 11 treatment groups with 5 repeats in each group and a control group. In the experimental group, the final concentration of Op-Meta was 40,50,6070 and 80 渭 g / mL, respectively. The cells were cultured for 24 h for 48 h and 72 h. The results showed that the whole activity of BMECs was the highest in the culture medium supplemented with different concentrations of Op-Met for 24 h. When the concentration of Op-Met was 60 渭 g / mL and the culture time was 24 hours, the expression of 伪 s1-casein gene (CSN1S1) and 尾 -casein gene (CSN2) increased significantly. The expression of PepTsgene and aminopeptidase nitrogen gene (APN) and the changes of AP content in and out of cells were measured. The cells were randomly divided into three treatment groups with 3 repeats in each group, one of which was a control group. The cells in the experimental group were cultured for 24 h with 60 渭 g / mL Met-Met or Met-Met-Met, respectively. The results showed that the expression of PepT2 and APN in the small peptide transport vector 2 (PepT2) and the expression of APN were significantly increased, and the AP content in the extracellular AP was significantly higher than that in the P0.01Met-Met Met-Met group (PepT1). And PepT2 expression increased significantly. The second part was based on the suitable F-Met concentration of 60 渭 g / mL in BMECs medium, which was determined by previous scholars. The expression of casein genes was determined by adding appropriate concentrations of F-MetMet-Met and Met-Met-Met in the culture of BMECs. The cells were randomly divided into four treatment groups with 3 repeats in each group and one as control group. In the experimental group, the cells were cultured at the appropriate concentration of F-Met-Met or Met-Met-Met for 24 h. The results showed that the addition of F-Met could significantly promote the expression of CSN1S1CSN2 and 魏 -casein gene (CSN3), the addition of Met-Met could significantly promote the expression of CSN1S1 (P0.01), and the addition of F-Met could significantly promote the expression of CSN1S1. In the third part, the expression of casein gene and its related genes and the content of AP in the BMECs of Op-Met were measured when replacing F-Met with different proportions. The cells were randomly divided into 10 treatment groups. Each group had 3 repetitions and one group was the control group. In the experimental group, 15% and 20%% Op-Met were added to the culture medium to replace F-Met.The results showed that the optimum substitution ratio of the F-Met was 15g when the Op-Met substitution was 0. The results showed that the addition of small peptide could increase the expression of BMECs casein gene. When the appropriate concentration of Op-Met was 60 渭 g / mL, CSN1S1 and CSN2 significantly increased the expression of PepT2 gene, and when Op-Met was used instead of 15F-Met, the expression of Op-Met casein gene was increased significantly. The expression of casein genes increased significantly.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823

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