| The aim of this study was to determine the effects of different supplementation concentrations of sodium acetate (0,6,9,12and15mmol/L) and sodium β-hydroxybutyrate(0,0.8,1.6and2.4mmol/L) and the ratios between them (1:1,2:1and4:1) on milk fat and protein synthesis in two-dimensional cultured bovine mammary epithelial cells (BMECs). The optimum concentration of sodium acetate and sodium β-hydroxybutyrate, as well as the optimum ratio of sodium acetate to sodium β-hydroxybutyrate, were selected for comparative study on the effects of short chain fatty acids on synthesis of milk fat and protein in two and three dimensional cultured BMECs. Meanwhile, the molecular mechanisms of short chain fatty acids regulation milk fat synthesis were clarified through genes expression of transcriptional regulation factors and genes associated with fatty acids and protein synthesis. Results of this thesis provided theory basis for directional regulation of milk fat synthesis. This study contained three trials.Trial one comprised two parts of content. In part one the effects of different concentration of sodium acetate(0,6,9,12and15mmol/L) on cell viability, triacylglycerol content (TAG), as well as mRNA expression of genes involved in milk fat and protein synthesis in two dimensional cultured BMECs were investigated. The results showed that TAG content in BMECs was significantly higher in group with addition of12mmol/L sodium acetate than in other groups (P<0.05). The mRNA expression of ACC, ACSS2, FAS, DGAT, CD36, PPARG, SREBP1, Akt-1and mTOR in BMECs were greater in groups with addition of12mmol/L and15mmol/L sodium acetate than in other groups. The mRNA expression of CSN1S1in BMECs were significantly higher in treatment with addition of6mmol/L sodium acetate than in treatments with supplementation of9mmol/L,12mmol/L and15mmol/L sodium acetate(P<0.05). In part two the optimum concentration of sodium acetate (12mmol/L) was used to comparative study the effects of sodium acetate on synthesis of milk fat and protein both in two and three dimensional cultured BMECs. The results showed that TAG content of cell culture medium in the three-dimensional culture condition were significantly higher than two-dimensional culture condition(P<0.05). as well as the mRNA expression of genes involved in milk fat and protein synthesis of BMECs were significantly higher than two-dimensional culture condition (P<0.001).In the treatment group, compared with the two-dimensional culture models, the mRNA expression of ACC, FAS, SREBP-1, PPARG,CSN1S1,CSN3were increased by170.74,97.39,96.9,149.52,160.33,109.15times, respectively. In the control group, compared with the two-dimensional culture models, the mRNA expression ofACC,FAS,SREBP-1, PPARG,CSN1S1,CSN3were increased by67.67,37.34,23.9,69.85,82.64,65.72times, respectively.Trial two was consisted of two parts of content. In part one the effects of different concentrations of sodium β-hydroxybutyrate (0,0.8,1.6and2.4mmol/L) on cell viability, TAG content, as well as mRNA expression of genes associated with milk fat and protein synthesis in two dimensional cultured BMECs were studied. The results showed that TAG content in BMECs was significantly higher in group with addition of2.4mmol/L sodium β-hydroxybutyrate than that of other groups (P<0.05). The mRNA expression of ACC, ACSS2, FAS and SREBP1were greater in groups with addition of1.6mmol/L and2.4mmol/L sodium β-hydroxybutyrate than those of in control group and group with supplementation of0.8mmol/L sodiumβ-hydroxybutyrate. However, for mRNA expression of DGAT, CD36,PPARG,Akt-1and mTOR the reverse tendency was observed, which were highest in group with addition of0.8mmol/L sodium β-hydroxybutyrate (P<0.05).The mRNA expression of CSN1S1and CSN3in BMECs were significantly lower in sodium β-hydroxybutyrate supplementation groups than that of in control group (P<0.05). In part two the optimum concentration of sodium β-hydroxybutyrate (2.4mmol/L) was used to comparative study the effects of sodium β-hydroxybutyrate on synthesis of milk fat and protein both in two and three dimensional cultured BMECs. The results showed that TAG content of cell culture medium in the three-dimensional culture condition were significantly higher than two-dimensional culture condition(P<0.05). as well as the mRNA expression of genes involved in milk fat and protein synthesis of BMECs were significantly higher than two-dimensional culture condition (P<0.001).In the treatment group, compared with the two-dimensional culture models, the mRNA expression of ACC, FAS, SREBP-1, PPARQCSN1S1were increased by142.63,56.29,118.56,31.69,51.71times, respectively.Trial three was composed of two parts of content. In part one the effects of different ratios between sodium acetate and sodium β-hydroxybutyrate (0,1:1,2:1and4:1) on cell viability, TAG content, as well as mRNA expression of genes involved in milk fat and protein synthesis in two dimensional cultured BMECs were studied The results showed that TAG content in BMECs was significantly higher in group of2:1and4:1than that of in control group and group of1:1(P<0.05). As compared with control group, mixed addition of sodium acetate and sodium β-hydroxybutyrate significantly up regulated the mRNA expression of genes involved in milk fat synthesis (P<0.05). In part two the optimum ratio between sodium acetate and sodium P-hydroxybutyrate (2:1) was used to comparative study the effects of mixed supplementation of sodium acetate and sodium β-hydroxybutyrate on synthesis of milk fat and protein both in two and three dimensional cultured BMECs. The results showed that TAG content of cell culture medium in the three-dimensional culture condition were significantly higher than two-dimensional culture condition(P<0.05). as well as the mRNA expression of genes involved in milk fat and protein synthesis of BMECs were significantly higher than two-dimensional culture condition (P<0.001).In the treatment group, compared with the two-dimensional culture models, the mRNA expression of ACC, FAS, SREBP-1, PPARG,CSN1S1were increased by179.25,53.25,107.31,213.72,144.75times, respectively.In summary, in the three-dimensional culture conditions, the promotion effect of short chain fatty acids on milk fat synthesis of BMECs were better in three cultured model than that of in two dimensional cultured model. |
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