| The aim of this study was to determine the effect of supplementation of leptin,insulinlike growth factor-I(IGF-I),hydrocortisone(HYD)and the different ratios of them on milk protein and milk fat synthesis in bovine mammary epithelial cells(BMECs).The suitable mode of the supplementation of hormone and cytokines for improving milk components synthesis and the molecular mechanism of milk fat and milk protein synthesis that regulated by hormone and cytokines were clarified through genes expression of transcriptional regulation factors and genes associated with milk fat and milk protein synthesis.Result of this thesis provided theory basis for the synthesis of milk fat and milk protein that regulated by endocrine system.This study contained four trials.The effect of different concentration of leptin(0、10、50、100、500 ng/m L),IGF-I(0、1、10、100、500 ng/mL),HYD(0、1、10、100、1000 ng/mL)and different ratios of them((0:0:0),(10:2:1),(2:2:1)and(200:1:100))on triglyceride content(TAG),as well as mRNA expression of genes involved in milk fat and protein synthesis were investigated in this four trails respectively.The group Ⅰwas control group which has no hormone and cytokines supplementation.The total quantity of hormone and cytokines for group Ⅱ,Ⅲ and were all 250 ng/mL..ⅣThe results of trial one showed that compared with group I,10~50 ng/mL leptin significantly upregulated m RNA expressions of S6K1,4EBP1 and CSN3(P<0.05).The m RNA expressions of AKT,m TOR,SREBP1 and DGAT2 were significantly higher in 50 ng/mL leptin group compared with those in control group(P<0.05).100 ng/mL leptin significantly upregulated the mRNA expression of SREBP1 and STAT5,500 ng/mL leptin significantly downregulated gene expression of 4EBP1 and DGAT2(P<0.05);all the dosage of leptin upregulated the gene expression of OB-Rb and PPARγ and the dosedependent effect was observed.All dosages of leptin supplementation have no significantly effect on the mRNA expression of ACC and FASN(P>0.05).when the dosage of the leptin over 50 ng/mL,The TAG and lipid droplet content was significantly lower than control group(P<0.05).The results suggest that 50 ng/mL leptin is optimal dosage,considering its simulative effects on protein synthesis,and the content of TAG and lipid droplet were significantly decreased by leptin.The results of trail two showed that all the dosages of IGF-I significantly upregulated the mRNA expression of IGF-IR,STAT5,AKT,mTOR,SREBP1 and PPARγ and the mRNA expression of STAT5,4EBP1 and SREBP1 were highest in the 100 ng/m L IGF-I group,the mRNA expression of IGF-IR,mTOR,AKT and PPARγ were highest in 500 ng/mL IGF-I group(P<0.05);10~500 ng/mL IGF-I treatment significantly upregulated the mRNA expression of S6K1 、 CSN3 and DGAT2 and the dose-dependent effect was observed(P<0.05).the highest mRNA expression of these three were in 500 ng/mL IGFI.The mRNA expression of ACC and the content of TAG and lipid droplet in 100~500 ng/mL IGF-I were significantly higher than those in control group(P<0.05);the mRNA expression of FASN was significantly upregulated by 100 ng/mL IGF-I(P<0.05).the results suggest that 100~500 ng/mL IGF-I have the optimal effect of promoting the synthesis of milk protein and milk fat.The result of trail three showed that 10~1 000 ng/mL HYD significantly upregulated the mRNA expression of GR,AKT and CSN3(P<0.05);compared with control group the mRNA expression of ACC and DGAT2 were significantly upregulated by 10~100 ng/mL HYD(P<0.05);The mRNA expression of PPARγ,SREBP1,FASN,m TOR,4EBP1,S6K1 and the content of TAG and lipid droplet were significantly higher than those in control group(P<0.05).These results suggest that 100 ng/mL HYD is the optimal dosage considering its simulative effect on synthesis of both milk protein and milk fat.The result of trail four showed that the mRNA expression of IGF-IR,GR,OB-Rb and genes involved in milk protein and milk fat synthesis except FASN,as well as the TAG content in 3D were significant higher than those in 2D when the IGF-I,HYD and leptin were added with different ratios(P<0.01).The mRNA expression of ACC,FASN,STAT5 and 4EBP1 in group Ⅱsignificantly higher than all the other groups and the m RNA expression and of PPARγ,SREBP1,CSN3 and TAG content were significantly upregulated also(P<0.05).The mRNA expression of IGF-IR and OB-Rb were the highest in Group Ⅲ.The mRNA expression of m TOR S6K1 and AKT were significantly upregulated by group Ⅳ(P<0.05).the mRNA expression of DGAT2 and the content of TAG were significantly decreased(P<0.05)in group Ⅲand group Ⅳthough they do upregulated the mRNA expression of SREBP1(P<0.01);the content of lipid droplet in group Ⅱ,Ⅲ,Ⅳwere significantly decreased compared with group Ⅰ(P<0.05).The results suggest that 3D cultured model is better than 2D when it comes to the effect of hormone and cytokine on BMECs in vitro.And the group is the optimal ratio of IGFⅡ-I,HYD and leptin considering its simulate effect on milk protein and milk fat synthesis. |
PDF Full Text Request