二、三维培养模式下添加激素对奶牛乳腺上皮细胞乳脂肪和乳蛋白合成的影响

发布时间：2018-08-23 09:30

【摘要】：本论文研究在二、三维培养模式下,添加不同浓度的催乳素(PRL)、胰岛素(INS)、生长激素(GH)及三者不同浓度配比对奶牛乳腺上皮细胞(BMECs)乳脂肪和乳蛋白合成的影响,从乳脂肪及乳蛋白合成相关基因和转录调控因子基因表达水平初步探明激素调控乳脂肪和乳蛋白合成的分子机制,为调控乳成分合成提供理论依据,为研究奶牛泌乳生物学机制时乳腺上皮细胞培养模型的选择提供数据支撑。论文包含四个试验,每个试验包含两部分研究内容,第一部分研究在二维培养模式下,单独添加PRL(0、100、300、500、1000ng/mL)、INS(0、25、100、400、800ng/mL)和 GH(0、10、30、100、300 ng/mL)以及组合添加不同配比的PRL、INS和GH[试验Ⅰ组(0)、试验Ⅱ组(20:1:2)、试验Ⅲ组(7.3:1:1.8)]研究其对BMECs甘油三酯(TAG)含量和乳脂肪及乳蛋白合成相关基因表达的影响。第二部分内容根据第一部分研究筛选出的适宜激素浓度进行单一或组合添加,同时进行BMECs的二维和三维培养。激素单一添加时PRL浓度为0、100和200 ng/mL,INS浓度为0、5和10 ng/mL,GH浓度为0、10和30 ng/mL。组合添加时PRL、INS和GH的总添加量相同,均为140 ng/mL,试验Ⅰ组为对照组、试验Ⅱ组三种激素的配比为10:1:3,试验Ⅲ组为20:1:2,试验Ⅳ组为2:1:1,比较不同培养模式下BMECs培养基中TAG含量以及乳脂肪和乳蛋白合成相关基因的表达差异。试验一研究表明,二维培养模式下,100 ng/mL PRL显著提高BMECs脂肪酸合成酶(FASNN)、雷帕霉素靶分子(mTOR)、核糖体p70s6激酶(S6K1)、真核翻译启始因子4E结合蛋白(4EBP1)、α-酪蛋白(CSN1S1)和催乳素受体(PRLR)基因的表达量。300ng/mL PRL显著提高乙酰辅酶A羧化酶a(ACC)、二脂酰甘油酰基转移酶2(DGAT2)、脂肪酸结合蛋白3(FABP3)、PRLR基因表达量及甘油三酯(TAG)的含量。结果提示,二维培养模式下100～300 ng/mL PRL促进乳脂肪和乳蛋白的合成,但是高浓度(1 000 ng/mL)的PRL对BMECs乳脂肪和乳蛋白的合成有一定抑制作用。三维培养模式下,培养基中TAG含量及BMECs乳脂肪和乳蛋白合成相关基因表达量均显著高于二维培养模式。100 ng/mL组AC FASN、硬脂酰CoA 去饱和酶(SCT)、DGAT2、固醇调节元件结合蛋白1(SREBP1)、过氧化物酶体增殖物激活受体γ(PPARG)、4EBP1、β-酪蛋白(CSN2)、K-酪蛋白(CSN3)、PRLR的mRNA相对表达量及TAG合成量显著高于对照组及200 ng/mL PRL组。200 ng/mL PRL组显著促进丝氨酸/苏氨酸激酶(AKT)、mTOR、CSN1S1基因表达量。结果表明,三维培养模式下PRL添加量为100 ng/mL时,对乳脂肪和乳蛋白的合成有较好的促进作用。试验二研究表明,在二维培养模式下INS浓度为25ng/mL和100ng/mL时,BMECs TAG含量显著高于对照组。25 ng/mL INS显著提高ACC、SREBP1基因表达量,100 ng/mL INS显著促进4EBP1基因表达量,显著抑制S6K1、ACC、EASN、SCD、DGAT2、SREBP1、FABP3、STAT5、ACSN3、INSR基因表达量。结果表明,二维培养模式下INS添加量为0～25 ng/mL时,对乳脂肪和乳蛋白的合成有较好的促进作用,100～800 ng/mL对乳脂肪和乳蛋白的合成有抑制作用。在三维培养模式下,培养基中TAG合成量及BMECs乳脂肪和乳蛋白合成相关基因表达量均显著高于二维培养模式。5 ng/mL INS显著上调ACC、PPARG、AKT、S6K1、CSN1S1 基因表达量;10 ng/mL INS 组 FASSN、DGAT2、FABP3、4EBP1、STAT5、IANSRR基因表达量及TAG合成量显著高于对照组及5 ng/mL组。结果表明,三维培养模式下INS添加量为10 ng/mL时,对乳脂肪和乳蛋白的合成有较好的促进作用。试验三研究表明,在二维培养模式下,10 ng/mL GH显著提高FASN、DGATT、SREBP1、PPARG、STAT5、CSN1S1、GHR基因表达量;30ng/mL GH显著提高SREBP1基因表达量及TAG合成量。结果表明,二维培养模式下GH添加量为10～30 ng/mL时,促进BMECs乳脂肪和乳蛋白的合成,100-300 ng/mL GH对BMECs乳脂肪和乳蛋白的合成有抑制作用。在三维培养模式下,培养基中TAG合成量及BMECs乳脂肪和乳蛋白合成相关基因表达量均显著高于二维培养模式。添加10 ng/mL GH显著上调BMECs内ACC、SCD、DGAT2、CSN2、CSN3基因表达量,显著抑制S6K1基因表达量;30 ng/mL组BMECs内FASN、SREBP1、PPARG、FABP3、AKT、mTOR、4EBP1、S6K1、STAT5、CSN1S1、GHR、IGFR基因表达量及TAG合成量显著高于对照组及10ng/mL组。结果表明,在三维培养模式下,GH添加量为30 ng/mL时,对BMECs乳脂肪和乳蛋白的合成有较好的促进作用。试验四研究表明,二维培养模式下,试验Ⅱ组(20:1:2)DGT、FABP3、SREBP1、CSN1S1、STAT5、mTOR、AKT、4EBP1、S6K1、GHR、IGFR基因表达量及 TAG 合成量显著高于对照组及试验Ⅲ组(7.3:1:1.8)。结果表明,二维培养模式下,PRL、INS和GH组合比例为20:1:2时,对BMECs乳脂肪和乳蛋白合成有较好的促进作用。在三维培养模式下,培养基中TAG合成量及BMECs乳脂肪和乳蛋白合成相关基因表达量均显著高于二维培养模式。试验Ⅱ组(10:1:3)ACC、FASNNN、DGAT1、FABP3、CSN1S1、CSN2、CSN3和AKT基因的表达量显著高于试验Ⅰ组、Ⅲ组(20:1:2)和Ⅳ组(2:1:1),SCDD、PPARG、mTOR、4EBP1、S6K1和STAT5基因的表达量显著高于试验Ⅰ组(P0.05)。试验Ⅱ组和Ⅲ组PRLR、INNSR、GHR和IGFR基因表达量显著高于试验Ⅰ组和Ⅳ组(P0.05)。试验Ⅲ组SCD和SREBP1基因表达量显著高于其他三组。试验Ⅳ组4EBP1、STAT5、IGFR基因的表达量显著高于其他三组。结果表明,三维培养模式下,PRL、INS和GH组合比例为10:1:3时,对BMECs乳脂肪和乳蛋白的合成有较好的促进作用。
[Abstract]:In this paper, the effects of different concentrations of prolactin (PRL), insulin (INS), growth hormone (GH) and their combinations on milk fat and milk protein synthesis in bovine mammary epithelial cells (BMECs) were studied under two-and three-dimensional culture mode. The expression levels of genes related to milk fat and milk protein synthesis and transcription regulators were preliminarily explored. To study the molecular mechanism of hormone regulating milk fat and protein synthesis and provide theoretical basis for regulating milk composition synthesis, and to provide data support for the selection of mammary gland epithelial cell culture model in studying the biological mechanism of lactation in dairy cows. Under the formula, PRL (0,100,300,500,1000ng/mL), INS (0,25,100,400,800ng/mL) and GH (0,10,30,100,300 ng/mL) were added separately and different ratios of PRL, INS and GH were added in combination [test group I (0), test group II (20:1:2), test group III (7.3:1:1.8)] to study the content of triglyceride (TAG) and the expression of genes related to milk fat and milk protein synthesis. In the second part, the optimum hormone concentration was selected according to the first part of the study. The two-dimensional and three-dimensional culture of BMECs was carried out simultaneously. The PRL concentration was 0,100 and 200 ng/mL, the INS concentration was 0,5 and 10 ng/mL, and the GH concentration was 0,10 and 30 ng/mL. The same, 140 ng / mL, test group I as the control group, test group II three hormones ratio of 10:1:3, test group III 20:1:2, test group IV 2:1:1, compare the different culture modes of BMECs medium TAG content and milk fat and milk protein synthesis related genes expression. Test 1 study, two-dimensional culture mode, 100 ng / 3 ML PRL significantly increased the expression of BMECs fatty acid synthase (FASNN), rapamycin target molecule (mTOR), ribosomal p70S6 kinase (S6K1), eukaryotic translation initiation factor 4E binding protein (4EBP1), alpha-casein (CSN1S1) and prolactin receptor (PRLR) genes. 300ng/mL PRL significantly increased the expression of ACA carboxylase (ACC), diacylglycerol transferase 2 (D-glycerol transferase 2). GAT2, FABP3, PRLR gene expression and triglyceride (TAG) content. The results suggested that 100-300 ng/mL PRL promoted the synthesis of milk fat and milk protein in two-dimensional culture mode, but high concentration (1 000 ng/mL) PRL inhibited the synthesis of milk fat and milk protein in BMECs. TAG content and BMECs milk fat and milk protein synthesis related gene expression were significantly higher than those of two-dimensional culture model. 100 ng/ml group AC FASN, stearoyl CoA desaturase (SCT), DGAT2, sterol regulatory element binding protein 1 (SREBP1), peroxisome proliferator-activated receptor gamma (PPARG), 4EBP1, beta-casein (CSN2), K-casein (CSN3), PRLR. The relative mRNA expression and TAG synthesis of PRL group were significantly higher than those of control group and 200 ng/mL PRL group. 200 ng/mL PRL group significantly promoted the expression of serine/threonine kinase (AKT), mTOR and CSN1S1 gene. The results showed that PRL supplementation of 100 ng/mL in three-dimensional culture mode could promote the synthesis of milk fat and milk protein. The results showed that the TAG content of BMECs was significantly higher than that of control group when INS concentration was 25 ng/mL and 100 ng/mL. 25 ng/mL INS significantly increased ACC, SREBP1 gene expression, 100 ng/mL INS significantly increased 4EBP1 gene expression, significantly inhibited S6K1, ACC, EASN, SCD, DGAT2, SREBP1, FABP3, STAT5, ACSN3, INSR gene expression. When the INS content was 0-25 ng/mL, it could promote the synthesis of milk fat and milk protein, and 100-800 ng/mL could inhibit the synthesis of milk fat and milk protein. 5 ng/mL INS significantly increased the expression of ACC, PPARG, AKT, S6K1, CSN1S1 genes; 10 ng/mL INS group FASSN, DGAT2, FABP3, 4EBP1, STAT5, IANSRR gene expression and TAG synthesis were significantly higher than the control group and 5 ng/mL group. Trial 3 showed that 10 ng/mL GH significantly increased the expression of FASN, DGATT, SREBP1, PPARG, STAT5, CSN1S1 and GHR genes under two-dimensional culture mode, and 30 ng/mL GH significantly increased the expression of SREBP1 gene and the synthesis of TAG. The results showed that the addition of 10-30 ng/mL GH promoted the expression of fat and milk protein in BMECs. In three-dimensional culture, TAG synthesis and expression of genes related to milk fat and milk protein synthesis in BMECs were significantly higher than those in two-dimensional culture. 10 ng/mL GH significantly increased the expression of ACC, SCD, DGAT2, CSN2 and CSN3 genes in BMECs. The expression of FASN, SREBP1, PPARG, FABP3, AKT, mTOR, 4EBP1, S6K1, STAT5, CSN1S1, GHR, IGFR and TAG synthesis in BMECs of 30 ng/mL group were significantly higher than those of control group and 10 ng/mL group. Experiment 4 showed that the expression of DGT, FABP3, SREBP1, CSN1S1, STAT5, mTOR, AKT, 4EBP1, S6K1, GHR, IGFR and TAG synthesis were significantly higher in group II (20:1:2) than in control group and group III (7.3:1:1.8). The results showed that the ratio of PRL, INS and GH was 20:1:2 for BMECs milk fat and fat. TAG synthesis and BMECs milk fat and milk protein synthesis related gene expression were significantly higher in three-dimensional culture medium than in two-dimensional culture model. ACC, FASNNN, DGAT1, FABP3, CSN1S1, CSN2, CSN 3 and AKT gene expression in test group II (10:1:3) were significantly higher than those in test group I and group III (20:1:3). The expression levels of PRLR, INNSR, GHR and IGFR genes in group II and group III were significantly higher than those in group I and group IV (P 0.05). The expression levels of SCD, PPARG, mTOR, 4EBP1, S6K 1 and STAT5 genes in group III were significantly higher than those in other three groups. The results showed that when the ratio of PRL, INS and GH was 10:1:3, the synthesis of milk fat and protein in BMECs was promoted.
【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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