二、三維培養(yǎng)模式下添加激素對(duì)奶牛乳腺上皮細(xì)胞乳脂肪和乳蛋白合成的影響
發(fā)布時(shí)間：2018-08-23 09:30
【摘要】：本論文研究在二、三維培養(yǎng)模式下,添加不同濃度的催乳素(PRL)、胰島素(INS)、生長激素(GH)及三者不同濃度配比對(duì)奶牛乳腺上皮細(xì)胞(BMECs)乳脂肪和乳蛋白合成的影響,從乳脂肪及乳蛋白合成相關(guān)基因和轉(zhuǎn)錄調(diào)控因子基因表達(dá)水平初步探明激素調(diào)控乳脂肪和乳蛋白合成的分子機(jī)制,為調(diào)控乳成分合成提供理論依據(jù),為研究奶牛泌乳生物學(xué)機(jī)制時(shí)乳腺上皮細(xì)胞培養(yǎng)模型的選擇提供數(shù)據(jù)支撐。論文包含四個(gè)試驗(yàn),每個(gè)試驗(yàn)包含兩部分研究內(nèi)容,第一部分研究在二維培養(yǎng)模式下,單獨(dú)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)以及組合添加不同配比的PRL、INS和GH[試驗(yàn)Ⅰ組(0)、試驗(yàn)Ⅱ組(20:1:2)、試驗(yàn)Ⅲ組(7.3:1:1.8)]研究其對(duì)BMECs甘油三酯(TAG)含量和乳脂肪及乳蛋白合成相關(guān)基因表達(dá)的影響。第二部分內(nèi)容根據(jù)第一部分研究篩選出的適宜激素濃度進(jìn)行單一或組合添加,同時(shí)進(jìn)行BMECs的二維和三維培養(yǎng)。激素單一添加時(shí)PRL濃度為0、100和200 ng/mL,INS濃度為0、5和10 ng/mL,GH濃度為0、10和30 ng/mL。組合添加時(shí)PRL、INS和GH的總添加量相同,均為140 ng/mL,試驗(yàn)Ⅰ組為對(duì)照組、試驗(yàn)Ⅱ組三種激素的配比為10:1:3,試驗(yàn)Ⅲ組為20:1:2,試驗(yàn)Ⅳ組為2:1:1,比較不同培養(yǎng)模式下BMECs培養(yǎng)基中TAG含量以及乳脂肪和乳蛋白合成相關(guān)基因的表達(dá)差異。試驗(yàn)一研究表明,二維培養(yǎng)模式下,100 ng/mL PRL顯著提高BMECs脂肪酸合成酶(FASNN)、雷帕霉素靶分子(mTOR)、核糖體p70s6激酶(S6K1)、真核翻譯啟始因子4E結(jié)合蛋白(4EBP1)、α-酪蛋白(CSN1S1)和催乳素受體(PRLR)基因的表達(dá)量。300ng/mL PRL顯著提高乙酰輔酶A羧化酶a(ACC)、二脂酰甘油�；D(zhuǎn)移酶2(DGAT2)、脂肪酸結(jié)合蛋白3(FABP3)、PRLR基因表達(dá)量及甘油三酯(TAG)的含量。結(jié)果提示,二維培養(yǎng)模式下100～300 ng/mL PRL促進(jìn)乳脂肪和乳蛋白的合成,但是高濃度(1 000 ng/mL)的PRL對(duì)BMECs乳脂肪和乳蛋白的合成有一定抑制作用。三維培養(yǎng)模式下,培養(yǎng)基中TAG含量及BMECs乳脂肪和乳蛋白合成相關(guān)基因表達(dá)量均顯著高于二維培養(yǎng)模式。100 ng/mL組AC FASN、硬脂酰CoA 去飽和酶(SCT)、DGAT2、固醇調(diào)節(jié)元件結(jié)合蛋白1(SREBP1)、過氧化物酶體增殖物激活受體γ(PPARG)、4EBP1、β-酪蛋白(CSN2)、K-酪蛋白(CSN3)、PRLR的mRNA相對(duì)表達(dá)量及TAG合成量顯著高于對(duì)照組及200 ng/mL PRL組。200 ng/mL PRL組顯著促進(jìn)絲氨酸/蘇氨酸激酶(AKT)、mTOR、CSN1S1基因表達(dá)量。結(jié)果表明,三維培養(yǎng)模式下PRL添加量為100 ng/mL時(shí),對(duì)乳脂肪和乳蛋白的合成有較好的促進(jìn)作用。試驗(yàn)二研究表明,在二維培養(yǎng)模式下INS濃度為25ng/mL和100ng/mL時(shí),BMECs TAG含量顯著高于對(duì)照組。25 ng/mL INS顯著提高ACC、SREBP1基因表達(dá)量,100 ng/mL INS顯著促進(jìn)4EBP1基因表達(dá)量,顯著抑制S6K1、ACC、EASN、SCD、DGAT2、SREBP1、FABP3、STAT5、ACSN3、INSR基因表達(dá)量。結(jié)果表明,二維培養(yǎng)模式下INS添加量為0～25 ng/mL時(shí),對(duì)乳脂肪和乳蛋白的合成有較好的促進(jìn)作用,100～800 ng/mL對(duì)乳脂肪和乳蛋白的合成有抑制作用。在三維培養(yǎng)模式下,培養(yǎng)基中TAG合成量及BMECs乳脂肪和乳蛋白合成相關(guān)基因表達(dá)量均顯著高于二維培養(yǎng)模式。5 ng/mL INS顯著上調(diào)ACC、PPARG、AKT、S6K1、CSN1S1 基因表達(dá)量;10 ng/mL INS 組 FASSN、DGAT2、FABP3、4EBP1、STAT5、IANSRR基因表達(dá)量及TAG合成量顯著高于對(duì)照組及5 ng/mL組。結(jié)果表明,三維培養(yǎng)模式下INS添加量為10 ng/mL時(shí),對(duì)乳脂肪和乳蛋白的合成有較好的促進(jìn)作用。試驗(yàn)三研究表明,在二維培養(yǎng)模式下,10 ng/mL GH顯著提高FASN、DGATT、SREBP1、PPARG、STAT5、CSN1S1、GHR基因表達(dá)量;30ng/mL GH顯著提高SREBP1基因表達(dá)量及TAG合成量。結(jié)果表明,二維培養(yǎng)模式下GH添加量為10～30 ng/mL時(shí),促進(jìn)BMECs乳脂肪和乳蛋白的合成,100-300 ng/mL GH對(duì)BMECs乳脂肪和乳蛋白的合成有抑制作用。在三維培養(yǎng)模式下,培養(yǎng)基中TAG合成量及BMECs乳脂肪和乳蛋白合成相關(guān)基因表達(dá)量均顯著高于二維培養(yǎng)模式。添加10 ng/mL GH顯著上調(diào)BMECs內(nèi)ACC、SCD、DGAT2、CSN2、CSN3基因表達(dá)量,顯著抑制S6K1基因表達(dá)量;30 ng/mL組BMECs內(nèi)FASN、SREBP1、PPARG、FABP3、AKT、mTOR、4EBP1、S6K1、STAT5、CSN1S1、GHR、IGFR基因表達(dá)量及TAG合成量顯著高于對(duì)照組及10ng/mL組。結(jié)果表明,在三維培養(yǎng)模式下,GH添加量為30 ng/mL時(shí),對(duì)BMECs乳脂肪和乳蛋白的合成有較好的促進(jìn)作用。試驗(yàn)四研究表明,二維培養(yǎng)模式下,試驗(yàn)Ⅱ組(20:1:2)DGT、FABP3、SREBP1、CSN1S1、STAT5、mTOR、AKT、4EBP1、S6K1、GHR、IGFR基因表達(dá)量及 TAG 合成量顯著高于對(duì)照組及試驗(yàn)Ⅲ組(7.3:1:1.8)。結(jié)果表明,二維培養(yǎng)模式下,PRL、INS和GH組合比例為20:1:2時(shí),對(duì)BMECs乳脂肪和乳蛋白合成有較好的促進(jìn)作用。在三維培養(yǎng)模式下,培養(yǎng)基中TAG合成量及BMECs乳脂肪和乳蛋白合成相關(guān)基因表達(dá)量均顯著高于二維培養(yǎng)模式。試驗(yàn)Ⅱ組(10:1:3)ACC、FASNNN、DGAT1、FABP3、CSN1S1、CSN2、CSN3和AKT基因的表達(dá)量顯著高于試驗(yàn)Ⅰ組、Ⅲ組(20:1:2)和Ⅳ組(2:1:1),SCDD、PPARG、mTOR、4EBP1、S6K1和STAT5基因的表達(dá)量顯著高于試驗(yàn)Ⅰ組(P0.05)。試驗(yàn)Ⅱ組和Ⅲ組PRLR、INNSR、GHR和IGFR基因表達(dá)量顯著高于試驗(yàn)Ⅰ組和Ⅳ組(P0.05)。試驗(yàn)Ⅲ組SCD和SREBP1基因表達(dá)量顯著高于其他三組。試驗(yàn)Ⅳ組4EBP1、STAT5、IGFR基因的表達(dá)量顯著高于其他三組。結(jié)果表明,三維培養(yǎng)模式下,PRL、INS和GH組合比例為10:1:3時(shí),對(duì)BMECs乳脂肪和乳蛋白的合成有較好的促進(jìn)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.
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S823
本文編號(hào)：2198632
[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.
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S823
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