| The development of the dairy industry is an important symbol of agricultural modernization.Milk is loved by humans for its rich proteins,fatty acids,minerals and vitamins.Milk protein and fat are the main components of milk andthe proportion of them is the determining factor of milk quality.The scale of China's dairy industry is gradually expanding,and milk production is also steadily increasing,but there is still a large gap in milk quality compared with developed countries.Proteins and fatty acids in milk are mainly secreted by bovine mammary epithelial cells,and regulated by related genes.All-trans retinoic acid?ATRA?has been shown to promote mammary gland development and increase milk protein secretion.In this study,we added all-trans retinoic acid during the differentiation of MAC-T cells to determine the effect of all-trans retinoic acid on the expression of milk casein and fatty acid-related genes and the milk fatty acid composition,in order to meet the expectancy of improving milk quality.Experiment 1:The adherent-grown MAC-T cells were seeded in 96-well plates at a density of 1×104 cells/ml,2×103 cellsper well.After 1 day of culture,each 96-well plate was divided into 9 groups of 6 replicates each.Added all-trans retinoic acid working solution to a concentration of 0.0,0.5,1.0,1.5,2.0,2.5,3.0,3.5,4.0?M,of which 0.0?M is added with0.004%dimethyl sulfoxide?The concentration of dimethyl sulfoxide in 4.0?M ATRA group?for 72 hours,and MTT assay was performed each 24 hours.The results showed that after treatment with ATRA,the proliferation activity of MAC-T cells increased first and then decreased with the increase of ATRA concentration.The most obvious concentration for promoting cell proliferation were 1.0?M in 1 day's group,1.5?M in 2 days'group and 1.0?M in 3 days'group.After two days of treatment,MAC-T proliferative activity was significantly inhibited when the ATRA concentration reached and exceeded 2.5?M,and significant inhibition occurred when the concentration reached 3.5?M after three days of treatment with ATRA.Experiment 2:Cells were seeded in 6-well plates at a density of 1×104 cells/ml.When the confluence rate reached about 80%,replace the medium with differentiation medium and cultured for 4 days.Then,the six-well plates were divided into 4 groups of 3 replicates each,and ATRA working solution was added at the final concentrations of 0.0,1.0,1.5,and 2.0?M,respectively.After cultured for another 3 days,the cells were collected and subjected to relevant gene detection,and phosphorylated protein detection at1.5?M ATRA were performed.The results showed that after treatment with ATRA,the?S1-casein and?-casein mRNA were increased,and ATRA also promoted the expression of JAK2,ELF5,S6K1 and4EBP1 mRNA expression,PI3K and eIF-4E were inhibited by ATRA,while STAT5-?,AKT1and mTOR mRNA expression were unaffected.on the other hand,STAT5-?and S6K1protein phosphorylation was enhanced when 1.5?M ATRA was treated,but mTOR was not affected.Experiment 3:Cells were seeded at a density of 1×104 cells/ml in100 mm culture dishes and 6-well plates.When the confluence rate reached about 80%,replaced the medium with differentiation medium and cultured for 4 days.Then,the 100 mm culture dishes and the6-well plates were divided into 4 groups,3 replicates in each group,ATRA working solution was added at the final concentrations of 0.0,1.0,1.5,and 2.0?M,respectively,and the cells were collected after 3 days,GC analysis and related gene detection were performed separately.The results showed that 1.5 and 2.0?M ATRA increased the content of short-chain fatty acids?SCFAs?and medium-chain fatty acids?MCFA?,while reducing the content of long-chain fatty acids?LCFA?.At the same time,all concentrations of ATRA increased the proportion of saturated fatty acids?SFAs?and monounsaturated fatty acids?MUFA?,decreased the proportion of polyunsaturated fatty acids?PUFA?;MUFA/SFA and PUFA/SFA ratios were also inhibited by ATRA.On the other hand,ATRA also reduced?-6 unsaturated fatty acids and increased?-3 unsaturated fatty acids,thereby significantly reducing the ratio of?-6/?-3.At the same time,ACACA,FASN,LPL,SCD,PPAR?and SREBP1 mRNA expression were all enhanced by ATRA.Conclusion:ATRA promotes casein gene expression by activating JAK2/STAT5 and the downstream signaling pathway of mTOR,increases the expression of milk fat synthesis-related genes?ACACA,LPL,FASN,SCD?by activating the SREBP1 signaling pathway to improve the fatty acid composition of MAC-T cells,by increases the proportion of short and medium chain fatty acids,monounsaturated fatty acids in cells,and increases EPA and DHA content,reduces the ratio of?-6/?-3. |
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