Effects Of Mthionine Oligopeptide On Expression Of Genes Involved In Casein Synthesis In Bovine Mammary Epithelial Cells

The thesis studied the effects of different concentrations of methionine oligopeptide(Op-Met)and Op-Met replacing different ratios of free methionine(F-Met)on expression of casein and its related genes and aminopeptidase(AP)content in bovine mammary epithelial cells(BMECs),establishes theoretical basis for revealing influence of oligopeptide on casein synthesis and its mechanism in BMECs.This research contains three parts:Two experiments were conducted in part one.In experiment one,the effects of different concentrations of methionine dipeptide(Met-Met)and methionine tripeptide(Met-Met-Met)on the expression of casein were studied under the condition of different culture times,to select the optimum culture time and concentration.The primary culture of BMECs was carried out by digestion method,and transferred to the 3rd generation.Cells were randomly assigned to 11 groups including a control group and 10 treatment groups,with 5 replicates in each treatment.Cells in experimental groups were treated with 40,50,60,70,and 80 ?g/mL Op-Met for 24 h,48 h,and 72 h.The results showed that:the overall cell viability was highest when cell were treated with Op-Met for 24 h;and expression of ?s1-casein(CSN1S1)and ?-casein(CSN2)increased significantly when cells were treated with 60?g/mL Op-Met for 24 h(P<0.05).Experiment two:under the optimum culture time and concentration of Op-Met,this experiment detected the expression of two peptide transporters and aminopeptidase N(APN),and intracellular and extracellular contents of AP.Cells were randomly assigned to 3 groups including a control group,each with 3 replicate.60 ?g/mL Met-Met or Met-Met-Met was added in culture medium and cultured cells for 24h.The results showed that:expression of peptide transporter 2(PepT2)and APN(P<0.05),and extracellular AP content(P<0.01)were increased by Met-Met treatment;expression of peptide transporter 1(PepT1)and PepT2 in Met-Met-Met group was improved(P<0.05).According to the optimum concentration of F-Met in BMECs determined by former researchers,60 ?g/mL F-Met was used in part two,to measure the effects of F-Met,Met-Met,and Met-Met-Met on abundance of casein genes in BMECs.Cells were randomly assigned to 4 groups including a control group,each with 3 replicate.Each experimental group contains proper concentration of F-Met,Met-Met,or Met-Met-Met and cultured for 24 h.The results showed that:addition of F-Met up-regulated expression of CSN1S1,CSN2,and ?-casein(CSN3)(P<0.01);addition of Met-Met enhanced abundance of CSN1S1(P<0.01);whereas expression of CSN3 was inhibited by addition of Met-Met-Met(P<0.01).Part three examined the variations of expression of casein and its related genes,and content of AP when Op-Met was replaced by different ratios of F-Met.Cells were randomly assigned to 10 groups including a control group,with 3 replicate in each group.In culture medium of treatment groups,0%,5%,10%,and 20%of Op-Met was added to replace F-Met and cultured cells for 24 h.The results showed that:the optimum ratio Op-Met replacing F-Met was 15%;when Op-Met ratio was 0%,extracellular AP content increased markedly(P<0.05).The results indicated that addition of oligo peptide of BMECs could stimulate expression of casein gene;60 ?g/mL Op-Met could increase expression of CSN1S1,CSN2 and PepT2;when 15%of F-Met was replaced by Op-Met,expression of casein related genes in cells was enhanced markedly.