硒对奶牛乳腺上皮细胞内乳脂肪和乳蛋白合成相关基因表达的影响

发布时间：2018-05-15 19:40

本文选题：乳脂肪 + 乳腺上皮细胞　；参考：《内蒙古农业大学》2017年硕士论文

【摘要】：本试验以奶牛乳腺上皮细胞(BMEC)为模型,以硒代蛋氨酸为原料,探究硒对BMEC抗氧化功能、乳脂肪和乳蛋白合成相关基因表达的影响。在此基础上进一步以脂多糖(LPS)为诱导应激源,研究硒对由LPS诱导损伤的BMEC的保护作用及其对乳脂肪和乳蛋白合成相关基因表达的影响,探讨了硒对LPS损伤情况下的BMECs是否具有保护作用,是否可减缓因LPS损伤弓引起的乳脂肪和乳蛋白合成的抑制作用。研究结果为科学改善奶牛乳腺氧化应激造成的产奶性能下降提供理论依据。本研究通过两部分试验进行,试验1采用单因子完全随机试验设计,研究了不同浓度的硒(0、10、20、50、100、150、200nmol/L)对BMEC活力、甘油三酯(TAG)含量、抗氧化相关酶活性、乳脂肪及乳蛋白合成相关基因及酶活性的影响。研究结果表明:硒对BMEC活力、谷胱甘肽过氧化物酶(GPX)、超氧化物歧化酶(SOD)活性及总抗氧化能力(T-AOC)的上调作用以及对丙二醛(MDA)浓度的下调作用呈显著的一次线性剂量依赖效应,其中以50～100nmol/L硒添加水平较好,150～200nmol/L硒添加水平促进效果有减弱的趋势。BMEC内TAG含量、脂肪酸合成酶(FASN)、硬脂酰辅酶A去饱和酶(SCD)、乙酰辅酶A羧化酶(ACACA)、脂蛋白酯酶(LPL)、雷帕霉素靶点(mTOR)酶活和p70核糖体蛋白S6激酶1(S6K1)的酶活性,受硒的影响不显著。硒对乳脂合成相关基因,ACACA、FASN、SCD、脂肪酸结合蛋白3(FABP3)、LPL、过氧化物酶体增殖物激活受体γ(PPARG)和固醇调节元件结合蛋白1(SREBF1)的基因表达水平均无显著的上调作用。硒对αs1-酪蛋白基因(CSN1S1)、K-酪蛋白(CSN3)、mTOR和信号转导及转录激活因子5(STAT5)、真核翻译起始因子4E结合蛋白1(4E-BP1)、真核翻译起始因子4E(eIF4E)、S6K1和酪氨酸激酶2(JAK2)等调控乳蛋白合成的相关基因的表达均无显著上调作用。综合得出50～100nmol/L的硒对健康BMECs的抗氧化功能具有较好的促进作用,但硒对健康BMECs的乳脂肪和乳蛋白的合成无显著影响。在试验1的基础上,试验2采用单因子完全随机试验设计,以LPS为应激源,将BMEC分为对照组(CON组)、LPS损伤组(LSO)和6个硒预保护组(LS10、LS20、LS50、LS100、LS150、LS200)。CON组在不含硒的细胞工作液中处理30h,不进行LPS处理:LS0组在不含硒的细胞工作液中处理24h,之后加入1μg/mL 的 LPS 处理 6h;硒预保护组 LS10、LS20、LS50、LS100、LS150、LS200,先分别用含10、20、50、100、200nmol/L的硒培养液培养24h,之后均加入1μg/mL的LPS处理6h。进一步探究硒对由LPS诱导损伤的BMEC的保护作用及其对乳脂肪和乳蛋白合成的影响。结果表明,LPS可以诱导BMEC产生氧化损伤,造成细胞活力和抗氧化功能的降低,引起GPX、SOD、CAT活性和T-AOC下降,MDA含量上升。硒对LPS诱导引起的BMEC的氧化损伤具有保护作用,引起上述指标呈相反变化,且保护作用与硒添加水平呈剂量依赖效应。LPS引起的BMEC氧化损伤可降低 ACACA、FASN、SCD、LPL、FABP3、SREBP1 和 PPARG 的基因表达及ACACA、FASN、SCD和LPL酶活性,抑制乳脂肪的合成;下调酪蛋白合成基因CSN1S1、CSN3的表达,降低了 mTOR和JAK/STAT两条信号通路的相关基因mTOR、STAT5、4E-BP1、eIF4E、S6K1 和 JAK2 的表达及 mTOR 和 S6K1 酶活性,抑制乳蛋白的合成。硒可有效减缓LPS诱导的BMEC损伤对乳脂肪与乳蛋白合成的抑制作用,引起上述指标的相反变化,且缓解作用与硒添加水平呈剂量依赖性,以50～200nmol/L的效果较好。综合以上结果,硒对由LPS诱导损伤后引起的BMEC的乳脂肪和乳蛋白合成及抗氧化功能的下降具有减缓作用,这与乳脂肪和乳蛋白合成相关基因的表达发生相应改变有关。
[Abstract]:In this experiment, the effect of selenium on BMEC antioxidant function, milk fat and milk protein synthesis related gene expression was studied with seleno methionine (BMEC) as the model. On this basis, the protective effect of selenium on BMEC induced by LPS and its effect on the milk fat induced by LPS were further studied. And the effect of gene expression related to milk protein synthesis, it is discussed whether selenium has protective effect on BMECs under LPS damage and whether it can slow down the inhibition effect of milk fat and milk protein synthesis caused by LPS injury. The results provide a theoretical basis for improving milk production performance caused by oxidative stress in dairy cows. Two experiments were carried out in two experiments. In Experiment 1, the effects of different concentrations of selenium (0,10,20,50100150200nmol/L) on the activity of BMEC, triglyceride (TAG), antioxidant related enzymes, milk fat and milk protein synthesis related genes and enzyme activities were studied. The results showed that selenium was active in BMEC, valley Cystamine peroxidase (GPX), superoxide dismutase (SOD) activity and total antioxidant capacity (T-AOC) up regulation and down regulation of malondialdehyde (MDA) concentration have a significant linear dose dependence effect, in which the addition level of 50 to 100nmol/L selenium is better, and the effect of 150 to 200nmol/L selenium supplementation has a weakening trend.B The contents of TAG in MEC, fatty acid synthetase (FASN), stearyl coenzyme A desaturase (SCD), acetyl coenzyme A carboxylase (ACACA), lipoprotein esterase (LPL), rapamycin target (mTOR) enzyme activity and p70 ribosomal protein S6 kinase 1 (S6K1) are not significantly affected by selenium. FABP3), LPL, peroxisome proliferator activated receptor gamma (PPARG) and sterol regulator element binding protein 1 (SREBF1) gene expression level have no significant up-regulation effect. Selenium on alpha s1- casein gene (CSN1S1), K- casein (CSN3), mTOR and signal transduction and transactionator 5 (STAT5), eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) The expression of eukaryotic translation initiation factor 4E (eIF4E), S6K1 and tyrosine kinase 2 (JAK2) had no significant up-regulation effect on the expression of related genes in milk protein synthesis. It was concluded that selenium from 50 to 100nmol/L had a good effect on the antioxidant function of healthy BMECs, but selenium had no significant effect on the synthesis of milk fat and milk protein in healthy BMECs. On the basis of test 1, test 2 was designed with single factor complete random test, and BMEC was divided into control group (group CON), LPS damage group (LSO) and 6 selenium pre protection groups (LS10, LS20, LS50, LS100, LS150, LS200), using LPS as the source of stress. 24h was treated with 1 mu g/mL, then 6h was added to LPS, and selenium pre protection group LS10, LS20, LS50, LS100, LS150, LS200 respectively. First, 24h was cultured respectively containing 10,20,50100200nmol/L selenium culture solution, then 1 micron treatment was added to further explore the protective effect of selenium on the induced injury and the synthesis of milk fat and milk protein. The results show that LPS can induce oxidative damage in BMEC, cause the decrease of cell vitality and antioxidant function, cause GPX, SOD, CAT activity and T-AOC decrease, MDA content increase. Selenium has protective effect on BMEC induced by LPS induced oxidative damage, causing the above indexes to be contrary, and the protective effect and selenium addition level are in a dose dependent manner. ACACA, FASN, SCD, LPL, FABP3, SREBP1 and PPARG could reduce the expression of ACACA, FASN, SCD, LPL, FABP3, SREBP1 and PPARG, and inhibit the synthesis of mammary fat by ACACA, FASN, SCD and inhibition, and down regulated the expression of the casein synthesis gene. The expression of JAK2 and the activity of mTOR and S6K1 enzyme inhibit the synthesis of milk protein. Selenium can effectively slow down the inhibitory effect of BMEC damage induced by LPS on the synthesis of milk fat and milk protein, and cause the reverse change of the above indexes, and the effect of selenium is dose-dependent with the level of selenium addition, and the effect of 50 to 200nmol/L is better. The above results, selenium synthesis results, selenium It has a slow effect on the decrease of milk fat and milk protein synthesis and the decrease of antioxidant function caused by LPS induced damage, which is related to the corresponding changes in the expression of milk fat and milk protein synthesis related genes.

【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

【参考文献】