【作者】 赵珂；

【导师】 刘建新；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2011， 博士

【摘要】 葡萄糖是乳汁合成的主要前体物质之一,与乳产量和乳品质密切相关。本研究围绕奶牛乳腺上皮细胞葡萄糖摄取的调控及其对乳成分合成的影响展开了以下研究：首先建立了泌乳奶牛乳腺上皮细胞模型；其次分别研究了泌乳相关激素和单糖基质对葡萄糖转运载体基因表达和葡萄糖摄取的影响；最后探讨了葡萄糖供应量对主要乳成分(乳脂和乳糖)合成的影响及其可能机制。主要研究结果如下：1.泌乳奶牛乳腺上皮细胞模型建立及其功能检测该部分旨在建立奶牛乳腺上皮细胞泌乳模型。试验选择DMEM/F-12培养液,用组织块培养法得到奶牛乳腺上皮细胞。为维持乳腺细胞的泌乳功能,在培养体系中添加了催乳素、胰岛素、氢化可的松、转铁蛋白等成分。根据乳腺上皮细胞和成纤维细胞对胰酶敏感度的不同,采用0.25%胰酶和0.15%胰酶加0.02%EDTA反复酶差消化,得到较为纯化的乳腺上皮样细胞。培养细胞呈单层、鹅卵石样铺展,可形成腺泡和岛屿状结构,具有上皮细胞的典型形态特征。此外,通过乳腺上皮细胞特征性细胞角蛋白18免疫细胞化学鉴定和细胞生长曲线分析,证明培养得到的细胞是正常乳腺上皮细胞。研究发现细胞传至第10代,细胞依然生长旺盛。通过细胞亚显微结构观察、乳脂形态观察、αsl酪蛋白基因表达、酪蛋白Western-blot含量检测等方法检测乳腺细胞的泌乳功能,证明培养至第10代的乳腺上皮细胞仍具有乳汁合成和分泌功能,表明本研究建立的奶牛乳腺上皮细胞培养模型可作为乳腺生理相关功能研究的模型。2.泌乳相关激素对乳腺细胞主要葡萄糖转运载体基因表达的影响及相关信号通路研究该部分研究了泌乳相关激素(催乳素、胰岛素和氢化可的松)单独及组合作用对GLUT1和GLUT8基因表达的影响,探讨了胰岛素调控GLUT8基因表达和葡萄糖摄取的相关信号通路。结果发现,不同浓度催乳素和胰岛素对GLUT1基因表达均无显著影响(P>0.05),高剂量催乳素可显著降低GLUT8基因表达(P<0.05)；添加胰岛素可促进GLUT8基因表达(P<0.05)；随着氢化可的松浓度增加,GLUT1和GLUT8基因表达均呈现先升高后降低的趋势。同时添加胰岛素(5 ng/mL)和氢化可的松,发现胰岛素和氢化可的松有明显的交互作用(P<0.01),氢化可的松可拮抗胰岛素提高GLUT8基因表达的作用；同时添加3种激素时,GLUT1和GLUT8基因表达与单独添加氢化可的松趋势一致。其次,利用胰岛素相关信号通路P13K、MAPK和PKC的抑制剂和激动剂,研究了胰岛素调控GLUT8基因表达和葡萄糖摄取的分子机理。研究发现,抑制MAPK对乳腺细胞葡萄糖摄取无显著影响(P>0.05),抑制PI3K可显著降低胰岛素促进的葡萄糖摄取和GLUT8基因表达(P<0.05),提示胰岛素可能主要通过P13K通路参与调控葡萄糖摄取;进一步研究PI3K的下游信号分子PKC发现,激活PKC可显著提高GLUT1、GLUT8基因表达和乳腺细胞葡萄糖摄取(P<0.05),抑制PKC可显著降低PKC激活所促进的GLUT1、GLUT8基因表达和葡萄糖摄取(P<0.05)。上述研究结果提示,GLUT1和GLUT8基因表达分别受不同泌乳相关激素的调控,胰岛素可能主要通过PI3K/PKC通路促进GLUT8表达和葡萄糖摄取。3.单糖基质和葡萄糖浓度对乳腺细胞糖摄取的调控及其机理研究该部分研究了不同类型单糖基质(葡萄糖或果糖)对乳腺细胞的影响以及葡萄糖浓度调控乳腺葡萄糖摄取的分子机制。首先,研究了葡萄糖或果糖对乳腺细胞生长、糖摄取和转运的影响。研究发现,添加葡萄糖或果糖组乳腺细胞活力均高于无糖组(P<0.05)；糖浓度相同时,果糖组的细胞活力显著高于葡萄糖组(P<0.05);随着葡萄糖或果糖浓度的增加,乳腺细胞活力均呈现先升高后降低的趋势,而糖摄取量显著升高(P<0.05)。上述研究结果提示,奶牛乳腺上皮细胞可以利用果糖,其转运和利用效率可能高于葡萄糖。其次,研究葡萄糖浓度对葡萄糖转运载体和己糖激酶的影响。结果发现,处理12 h时,与对照组(2.5 mmol/L)相比,5或10 mmol/L葡萄糖不影响GLUT1 mRNA丰度(P>0.05),而20 mmol/L葡萄糖可显著降低GLUT1基因表达(P<0.05),高浓度葡萄糖组(5、10和20 mmol/L)的己糖激酶酶活均高于对照组(P<0.05),而不同葡萄糖浓度对GLUT8和HK2的mRNA丰度均无显著影响(P>0.05);处理24时,GLUT1、GLUT8和HK2基因表达均随着葡萄糖浓度的增加而降低(P<0.05)；添加HK2抑制剂三-溴丙酮酸(3-BrPA)可显著降低2.5和10 mmol/L葡萄糖组的葡萄糖摄取和细胞活力(P<0.05)。研究结果提示,葡萄糖浓度可能部分通过提高HKs酶活调控乳腺细胞葡萄糖摄取,而HK2在此过程中起重要作用。4.葡萄糖浓度对乳腺细胞主要乳成分合成和葡萄糖代谢的影响该部分研究了葡萄糖供应对奶牛乳腺细胞主要乳成分合成和葡萄糖代谢的影响。研究发现,不同浓度葡萄糖(5、10或20 mmol/L)可影响乳脂合成关键酶的基因表达,处理12时,与5 mmol/L葡萄糖组相比,增加葡萄糖浓度对乙酰CoA羧化酶(ACC)、脂肪酸合成酶(FAS)、二酰甘油酰基转移酶(DGAT)和三磷酸甘油酰基转移酶(GPAT)的基因表达量均无显著影响(P>005)；处理24和48 h时,ACC、FAS、DGAT和GPAT的基因表达量随葡萄糖浓度的增加而降低(P<005),但均高于相同葡萄糖浓度处理12h组。其次,研究当葡萄糖供应充足时(处理12 h)葡萄糖浓度对乳脂含量的影响,结果发现,乳腺细胞甘油三酯含量随葡萄糖浓度的增加而增加,其中10和20 mmol/L组显著高于2.5mmol/L组(P<005)。不同浓度葡萄糖处理12 h对乳糖合成、糖酵解和磷酸戊糖途径的影响。结果发现,与对照组(2.5 mmol/L)相比,高浓度葡萄糖(5和10 mmol/L)可显著提高β-1,4-半乳糖基转移酶(B4GALT)的基因表达量(P<005)；不同浓度葡萄糖不影响乳清蛋白(LA)基因表达量(P>005)；10和20 mmol/L葡萄糖可显著提高乳糖合成酶蛋白含量(P<005)；10 mmol/L组丙酮酸激酶和葡萄糖6磷酸脱氢酶活力均显著高于2.5 mmol/L组；高浓度葡萄糖组(10 mmol/L)的线粒体势能和细胞ATP含量均高于低浓度葡萄糖组(2.5 mmol/L)(P<005)。上述研究结果提示,增加葡萄糖供应可能增加了乳糖合成底物浓度,促进乳糖合成,促进葡萄糖酵解和磷酸戊糖途径的代谢,增加细胞能量产出,进而提高乳腺乳脂合成量。综上所述,本研究成功构建了泌乳奶牛乳腺上皮细胞模型,利用此模型研究发现,奶牛乳腺上皮细胞葡萄糖摄取受泌乳相关激素、单糖基质类型和浓度的影响。其中胰岛素在奶牛乳腺葡萄糖摄取中发挥重要作用,它可能通过PI3K/PKC通路影响GLUT8表达从而调控葡萄糖摄取；葡萄糖浓度对葡萄糖摄取的调控主要通过影响HKs活力而不是主要葡萄糖转运载体基因表达而实现；葡萄糖浓度可影响乳成分合成,增加葡萄糖供应可能通过调控乳腺葡萄糖代谢而促进主要乳成分如乳脂和乳糖的合成。更多还原

【Abstract】 This study were conducted to investigate the regulation of glucose uptake and milk synthesis in bovine mammary epithelial cells (BMEC). Firstly, a lactating BMEC model was established; then the effects of lactogenic hormones and monosaccharide substrates on the gene expression of glucose transporters (GLUTs) and glucose uptake were determined; finally, the effects of glucose availability on milk synthesis and the possible mechanism was studied.1.Establishment and characterization of lactating BMEC modelThe objective of this study was to establish an in vitro lactating BMEC model. Mammary tissues were dispersed and cultured in DMEM/F12 medium containing insulin, prolactin, hydrocortisone, transferrin, epidermal growth factor and fetal calf serum. After the cells migrating from the tissue reach-80% of confluency, the tissues were removed and secretory epithelial cells were enriched by digesting with 0.25% trypsin repeatly to remove fibroblasts. The BMEC displayed a monolayer, cobblestone, epithelial-like morphology, and formed alveoli-like structures and island monolayer aggregates. The isolated cells were identified as epithelial origin by staining with antibody against cytokeratine 18. A one-half logarithmically growth curve, abundant microvilli, cytoplasmic lipid droplets, transcription of asl casein gene and synthesis of as caseins were observed. Thus, the lactating BMEC model can be an effective model in vitro for studies of milk synthesis of bovine mammary gland.2. The study on the regulation of glucose transporters gene expression and signaling pathwayThe study was conducted to determine the effects of lactogenic hormones (prolactin, insulin and hydrocortisone) on expression of GLUTs gene and the possible signaling pathway involved in the process in BMEC. The results showed that inclusion of prolactin and insulin did not affect abundance of GLUT1 mRNA (P> 0.05); prolactin at high down-regulated, whereas insulin at all concentrations up-regulated the expression of GLUT8 mRNA (P< 0.05), indicating that GLUT8 may be responsive to insulin in BMEC; the abundance of GLUT1 and GLUT8 mRNA increased by low concentration of hydrocortisone, but decreased when treated at higher level. Interaction of hydrocortisone and insulin on GLUT8 gene expression was observed (P< 0.01), and hydrocortisone counteract the insulin-stimulated expression of GLUT8 mRNA. When three hormones were added together at their physiological concentrations during lactation (100 ng/mL prolactin,5 ng/mL insulin and 100 ng/mL hydrocortisone), the expression of GLUT1 and GLUT8 mRNA was depressed (P< 0.05), similar to the result with 100 ng/ml of hydrocortisone. It is inferred that lactogenic hormones may not be involved in the regulation of abruptly increased expression of GLUT1 and GLUT8 mRNA in bovine mammary gland during the early lactation. Pretreatment with SB203580, an inhibitor of p-38 MAPK, did not influence the insulin-induced glucose uptake (P> 0.05). In contrast, LY294002, a specific inhibitor of PI3-K, for 30 min, significantly reduced the insulin-stimulated glucose uptake (P< 0.05). PKC is the downstream effector of PI3K, and addition of PMA (50 and 100 ng/mL), the agonist of PKC, stimulated the gene expression of GLUT1/GLUT8 and glucose uptake (P< 0.05). Furthermore, addition of PKC inhibitor (GF1090203X) (0.5,1 or 2μM), decreased the GLUT1/ GLUT8 gene expression and glucose uptake stimulated by PMA (P< 0.05). These results indicate that insulin may stimulate glucose uptake primarily via PI3K/PKC linked signaling pathways.3. The study on mechanism of glucose transport regulated by monosaccharide substrate and glucose availability in BMECGlucose and fructose were used to investigate the effect and mechanism of monosaccharide on cell viability and glucose transport by BMEC. Compared with the group without any monosaccharide, addition of glucose or fructose (5 or 10 mmol/L) increased the cell viability (P < 0.05), and the cell viability was higher with fructose than that with the same concentration of glucose (P< 0.05); viability of cells increased firstly and then decreased with increasing concentration of glucose/fructose, with the highest value at 10 mmol/L (P< 0.05); the uptake of each monosaccharide increased with increasing concentration of glucose/fructose, with the largest uptake at 20 mmol/L (P< 0.05). The plasma of mammalians has only few of fructose, and then we focus on the glucose transport in the following experiments. When incubated for 12 h, compared with control (2.5 mmol/L),5 and 10 mmol/L glucose did not influence abundance of GLUT1 mRNA (P< 0.05), while the expression of GLUT8 mRNA was not affected by any concentration of glucose (P> 0.05). For 24 h, GLUT1 and GLUT8 gene expression decreased with increasing concentration of glucose (P< 0.05), and the GLUT1 and GLUT8 gene expression at all concentrations of glucose was higher than their counterparts for 12 h (P< 0.05), except for at 20 mmol/L glucose. The HKs activity at high concentration of glucose (5,10 or 20 mmol/L) was higher than that at 2.5 mmol/L (P< 0.05). The expression of HK2 other than HK1 mRNA was detected in the BMEC, and abundance of HK2 mRNA was not affected by any concentration of glucose compared with 2.5 mmol/L glucose for 12 h (P< 0.05); the expression of HK2 gene decreased with increasing concentration of glucose for 24 h and had no difference with that of their counterpart for 12 h (P> 0.05). Furthermore, addition of 3-bromopyruvate (3-BrPA), the inhibitor of HK2, resulted in the decrease of glucose uptake and cell viability at both 2.5 and 10 mM glucose (P< 0.05), respectively. Therefore, the glucose concentrations might affect glucose uptake partly by altering activity of HKs, and HK2 might play important role in the process.4. Effect of glucose availability on milk synthesis and glucose metabolism in BMECThe BMEC were used to investigate the effect of glucose availability on milk synthesis and glucose metabolism. When treated for 12 h, compared with 5 mmol/L glucose, the increasing concentration of glucose (10 or 20 mmol/L) did not affect the mRNA abundance of acety-CoA carboxylase (ACC), fatty acid synthase (FAS), diacyl glycerol acyl transferase (DGAT) and glycerol-3 phosphate acyl transferase (GPAT) gene (P< 0.05); for 24 h, the mRNA abundance of ACC, FAS, DGAT and GPAT gene decreased with increasing concentration of glucose (P< 0.05). The expression profile of sterol regulatory element binding protein-1 (SREBP-1) showed the same pattern with ACC, FAS, DGAT and GPAT cultured with different concentrations of glucose. In addition, the content of triglyceride in the BMEC was higher with the high glucose availability group than that with lower one for 12 h (P< 0.05).The effects of glucose availability (2.5,5,10 or 20 mmol/L) on lactose synthesis and glucose metabolism for 12 h were also investigated. The gene expression of beta-1,4-galactosyl transferase (B4GALT) increased firstly and decreased lately with increasing concentration of glucose, and the gene expression ofα-lactalbumin (LA) was not affected by any concentrations. While the content of lactose synthase increased with increasing concentration of glucose, with the highest value at 20 mmol/L. Besides, the increased glucose concentration stimulated the activity of pyruvate kinase (PK) and glucose-6-phosphate dehydrogenase (G6PH), and elevated the energy status of the BMEC (P< 0.05).In summary, a lactating BMEC model was established, and it is found that the glucose metabolism may be regulated by Iactogenic hormones. Insulin plays important role in regulation of glucose uptake in BMEC and it may work by influencing the GLUT8 expression through PI3K/PKC signaling pathway. Glucose avalibility may affect the glucose transport mainly by altering activity of HKs and increasing glucose availaility may stimulate the milk fat and lactsoe synthesis by affecting the glucose metabolism in the BMEC.更多还原