發(fā)布時間：2018-05-15 19:40

  本文選題：乳脂肪 + 乳腺上皮細胞　； 參考：《內蒙古農業(yè)大學》2017年碩士論文

【摘要】：本試驗以奶牛乳腺上皮細胞(BMEC)為模型,以硒代蛋氨酸為原料,探究硒對BMEC抗氧化功能、乳脂肪和乳蛋白合成相關基因表達的影響。在此基礎上進一步以脂多糖(LPS)為誘導應激源,研究硒對由LPS誘導損傷的BMEC的保護作用及其對乳脂肪和乳蛋白合成相關基因表達的影響,探討了硒對LPS損傷情況下的BMECs是否具有保護作用,是否可減緩因LPS損傷弓引起的乳脂肪和乳蛋白合成的抑制作用。研究結果為科學改善奶牛乳腺氧化應激造成的產奶性能下降提供理論依據。本研究通過兩部分試驗進行,試驗1采用單因子完全隨機試驗設計,研究了不同濃度的硒(0、10、20、50、100、150、200nmol/L)對BMEC活力、甘油三酯(TAG)含量、抗氧化相關酶活性、乳脂肪及乳蛋白合成相關基因及酶活性的影響。研究結果表明:硒對BMEC活力、谷胱甘肽過氧化物酶(GPX)、超氧化物歧化酶(SOD)活性及總抗氧化能力(T-AOC)的上調作用以及對丙二醛(MDA)濃度的下調作用呈顯著的一次線性劑量依賴效應,其中以50～100nmol/L硒添加水平較好,150～200nmol/L硒添加水平促進效果有減弱的趨勢。BMEC內TAG含量、脂肪酸合成酶(FASN)、硬脂酰輔酶A去飽和酶(SCD)、乙酰輔酶A羧化酶(ACACA)、脂蛋白酯酶(LPL)、雷帕霉素靶點(mTOR)酶活和p70核糖體蛋白S6激酶1(S6K1)的酶活性,受硒的影響不顯著。硒對乳脂合成相關基因,ACACA、FASN、SCD、脂肪酸結合蛋白3(FABP3)、LPL、過氧化物酶體增殖物激活受體γ(PPARG)和固醇調節(jié)元件結合蛋白1(SREBF1)的基因表達水平均無顯著的上調作用。硒對αs1-酪蛋白基因(CSN1S1)、K-酪蛋白(CSN3)、mTOR和信號轉導及轉錄激活因子5(STAT5)、真核翻譯起始因子4E結合蛋白1(4E-BP1)、真核翻譯起始因子4E(eIF4E)、S6K1和酪氨酸激酶2(JAK2)等調控乳蛋白合成的相關基因的表達均無顯著上調作用。綜合得出50～100nmol/L的硒對健康BMECs的抗氧化功能具有較好的促進作用,但硒對健康BMECs的乳脂肪和乳蛋白的合成無顯著影響。在試驗1的基礎上,試驗2采用單因子完全隨機試驗設計,以LPS為應激源,將BMEC分為對照組(CON組)、LPS損傷組(LSO)和6個硒預保護組(LS10、LS20、LS50、LS100、LS150、LS200)。CON組在不含硒的細胞工作液中處理30h,不進行LPS處理:LS0組在不含硒的細胞工作液中處理24h,之后加入1μg/mL 的 LPS 處理 6h;硒預保護組 LS10、LS20、LS50、LS100、LS150、LS200,先分別用含10、20、50、100、200nmol/L的硒培養(yǎng)液培養(yǎng)24h,之后均加入1μg/mL的LPS處理6h。進一步探究硒對由LPS誘導損傷的BMEC的保護作用及其對乳脂肪和乳蛋白合成的影響。結果表明,LPS可以誘導BMEC產生氧化損傷,造成細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。硒對LPS誘導引起的BMEC的氧化損傷具有保護作用,引起上述指標呈相反變化,且保護作用與硒添加水平呈劑量依賴效應。LPS引起的BMEC氧化損傷可降低 ACACA、FASN、SCD、LPL、FABP3、SREBP1 和 PPARG 的基因表達及ACACA、FASN、SCD和LPL酶活性,抑制乳脂肪的合成;下調酪蛋白合成基因CSN1S1、CSN3的表達,降低了 mTOR和JAK/STAT兩條信號通路的相關基因mTOR、STAT5、4E-BP1、eIF4E、S6K1 和 JAK2 的表達及 mTOR 和 S6K1 酶活性,抑制乳蛋白的合成。硒可有效減緩LPS誘導的BMEC損傷對乳脂肪與乳蛋白合成的抑制作用,引起上述指標的相反變化,且緩解作用與硒添加水平呈劑量依賴性,以50～200nmol/L的效果較好。綜合以上結果,硒對由LPS誘導損傷后引起的BMEC的乳脂肪和乳蛋白合成及抗氧化功能的下降具有減緩作用,這與乳脂肪和乳蛋白合成相關基因的表達發(fā)生相應改變有關。
[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.

【學位授予單位】：內蒙古農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S823

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