维生素A和乙酸对奶牛乳腺上皮细胞乳脂乳蛋白合成相关基因表达的影响

发布时间：2018-03-03 05:00

本文选题：维生素A　切入点：乙酸　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】：本试验旨在研究维生素A(V_A)对奶牛乳腺上皮细胞(BMECs)内乳脂乳蛋白合成相关基因表达的影响,并在此基础上添加乙酸,进而观察在补充了乳脂肪合成前体物的条件下,二者对乳成分合成的影响是否存在互作关系,以期能深入了解V_A对乳脂乳蛋白合成的影响机制,为奶牛生产中V_A的合理添加以及改善乳脂乳蛋白的合成提供依据。试验一采用单因子完全随机试验设计,将贴壁生长的第三代BMECs以2×10~5个/mL的密度接种于6孔板中继续培养,待其贴壁率达到80%左右时随机分为6个处理组,每组6个重复。使用无血清培养基饥饿24 h后,添加V_A浓度分别为0、0.05、0.1、0.2、1.0 和 2.0μg/mL 的细胞培养液,0μg/mLV_A 为对照组,继续培养24h后收集细胞,进行相关指标的测定。试验结果表明:].0、2.0μg/mL V_A可以显著提高BMECs细胞活力以及甘油三酯(TG)的合成;V_A能上调乳脂肪合成调控因子过氧化酶体增殖物激活受体(PPARG)、固醇调节元件结合蛋白1(SREBP1)、硬脂酰辅酶A去饱和酶(SCD)的基因表达;V_A也能促进信号转导转录激活因子5(STAT5)、αsl-酪蛋白(CSN1S1)、K-酪蛋白(CSN3)等乳蛋白合成相关基因的相对表达。试验二采用二因素完全随机试验设计,将贴壁生长的第三代BMECs随机分为6个处理组,每组6个重复。使用无血清培养基饥饿处理24 h后,分别添加不同V_A浓度和乙酸浓度(乙酸钠为原料)的工作液,6种工作液的浓度分别是:0μg/mLV_A+0 mmol/L 乙酸,0μg/mL V_A+6 mmol/L 乙酸,0 μg/mL V_A+10 mmol/L乙酸,1 μg/mLV_A+Ommol/L 乙酸,1 μg/mL V_A+6 mmol/L 乙酸,1 μg/mLV_A+10 mmol/L乙酸;0μg/mLV_A+O mmol/L乙酸为对照组。继续培养24 h后按照测定指标要求的方法进行细胞收集和相关指标的测定。试验结果表明:V_A和乙酸在乳脂合成方面存在互作效应,乙酸能提高BMECs内的TG含量,但同时添加V_A则抑制TG合成;乙酸能上调BMECs内乙酰辅酶A羧化酶(ACACA)、脂肪酸合成酶(FASN)的活性及PPARG基因的表达,同时添加V_A则起抑制作用;乙酸能显著提高BMECs内哺乳动物雷帕霉素靶蛋白(mTOR)含量、S6K1酶活性,以及上调CSN3基因的表达,V_A与乙酸在mTOR与CSN2的基因表达方面具有互作效应,二者同时添加会减弱单一添加V_A或乙酸时对mTOR与CSN2的基因表达的下调作用。综合考虑,0V_A+10乙酸组对乳脂合成的促进作用较好,1V_A+10乙酸组较弱;OV_A+6乙酸组对乳蛋白合成促进作用较好,1V_A+6乙酸组较弱。
[Abstract]:The purpose of this study was to investigate the effect of vitamin A / V _ (V _ (A)) on the expression of genes related to milk fat milk protein synthesis in milk epithelial cells (BMECs) of dairy cows, and to add acetic acid on the basis of this, and then observe the condition of supplementation of precursor of milk fat synthesis. Whether there is an interaction between the two effects on the synthesis of milk ingredients, in order to understand the mechanism of the effect of VSAT on the synthesis of milk milk protein, In experiment 1, the third generation BMECs of adherent growth was inoculated in 6 hole plate with density of 2 脳 10 ~ 5 / mL, and the third generation of BMECs was inoculated in 6 hole plate with the density of 2 脳 10 ~ 5 / mL. When the adherence rate was about 80%, each group was randomly divided into six treatment groups, each group was subjected to 6 replicas. After starvation in serum-free medium for 24 hours, the cells were collected after 24 hours of starvation with 0 0. 05 渭 g / mL and 2. 0 渭 g / mL of Va, respectively. The results showed that:] 0 渭 g / mL Vapa could significantly increase the viability of BMECs cells and the synthesis of triglyceride (TG) could up-regulate the expression of lipid synthesis regulator, peroxisome proliferator activator receptor (PPARGN) and steroid regulator. The gene expression of stearyl coenzyme A desaturase (SCD1) could also promote the relative expression of signal transduction activator 5 (STAT5), 伪 sl-casein 1 (CSN1S1) K- casein casein (CSN3) and other genes related to milk protein synthesis. Experiment 2 used two factors to study the expression of genes related to milk protein synthesis, such as protein binding protein 1 (SREBP1) and stearyl coenzyme A desaturase (SCD). Complete random trial design, The third generation BMECs of adherent growth was randomly divided into 6 treatment groups with 6 replicates in each group. The concentration of six kinds of working liquid with different VA concentration and different concentration of acetic acid (sodium acetate as raw material) were 1: 0 渭 g / mLVA0 mmol/L, 0 渭 g / mLVA6 mmol/L acetic acid = 0 渭 g / mLVA 10 mmol/L acetic acid, 1 渭 g / mLVA Ommol/L acetic acid 1 渭 g / mLVA Ommol/L Ommol/L 1 渭 g / mLVA / 6 mmol/L / mmol/L, 1 渭 g / mLVA = 10 渭 g / mLVA = 10 渭 g / mLVA = 10 渭 g / mLVA = 1 渭 g / mLVA = 1 渭 g / mLVA = 1 渭 g / mLVA10. Mmol/L acetic acid 0 渭 g / mLVA O mmol/L acetic acid was used as the control group. After 24 hours of culture, cell collection and related indexes were determined according to the method required for the determination of the indexes. The results showed that there was an interaction effect between the two groups in the synthesis of milk fat. Acetic acid could increase TG content in BMECs, but at the same time add VA to inhibit TG synthesis, acetic acid could upregulate the activity of acetyl coA carboxylase, fatty acid synthase (FASN) and the expression of PPARG gene in BMECs. Acetic acid could significantly increase the activity of S6K1 enzyme in mammalian rapamycin target protein mTORand up-regulate the expression of CSN3 gene in BMECs. VA and acetic acid could interact with each other in the gene expression of mTOR and CSN2. The down-regulation of gene expression of mTOR and CSN2 was weakened by the addition of both of them. Considering the effect of VA10 acetic acid on the synthesis of milk fat, the effect of VA10 acetic acid on milk fat synthesis was better. 1 VA10 acetic acid group was weaker than OVA6 acetic acid group on milk protein. The effect of synthesis was better than that of 1 V A 6 acetic acid group.
【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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