| the subject make the cow mammary tissue for the material of culturing the mammary epithelial cell; and on the basis, collocated casein nutrient medium to study that how small peptides influence on the milk protein synthesis of mammary epithelial cells.1. Identification of the biological characteristics of mammary epithelial cellsCultured mammary epithelial cells by collagenase digestion, cell morphology was observed on the forth day of the primary and on the second day of the passage, drew the cell standard curves and cytoskeletal proteins-keratin18(cytokeratin-18)identification, the results indicate that the cells have the biological characteristics of breast epithelial cells, and can be used for continued testing.2. Three kinds of small peptides impacted on the proliferation of mammary epithelial cellsBased on casein model, the equal quality of met-met replaced the free met and1/2times the free met in medium, which were considered as the group A and the group B; so phe-phe did, and which were considered as the group C and the group D; subject to the matter of the material quality of free met, the equal material quality of free met were replaced respectively by0.007mmol met-lys and0.0134mmol met-lys, as the group E and the group F, free lys made up for inadequate lys in solution, other free amino acids in the same and additives were put into the media except serum. The mammary epithelial cells were collected and were inoculated into96-well plates to the density of5*104/ml, treated group were treated with casein medium which included small peptides, and the control group were treated with casein medium without small peptides, there were three replicates per hole. The OD value of The cells were detected in the absorbance for the nest460in24h,48h,72h, and the rate of the cell proliferation was detected.The results showed that the treated groups inhibited cell proliferation when the cells were cultured in the board for24h,A was significant different with the control group(p<0.05), E and F were respectively significantly different with the control group (p<0.05); in48h, treated ones inhibited cell proliferation, there were not the significant differences between the treatment groups, But, respectively, they were significant different from the control one(p<0.05);72h after,A and B promoted cell proliferation, and other treats inhibited cell proliferation, C、D and the control group were significantly different(p<0.05), E、F were respectively highly different (p<0.0001).3. Some peptides impacted on the genes expression of important protein and secretion of major caseinsAs same as the experiment2,cells were collected and inoculated into six-well plates with a density of2*105/ml, there were three replicates each treatment. After48h, RNA was extracted and determined to build great RT-PCR Detection system,which can detect the gene expression of as1、 as2、 β、k-casein and beta-lactoglobulin; elisa kit the secretion of major caseins to study the impact of small peptides on milk protein synthesis.Met-met on milk protein synthesis:the difference of the CSN1S2expression was not significant among the treated groups and the control group, A improved the expression of the CSN1S2, B inhibited the expression of the CSN1S2; the level of the LGB gene expression was significant different(p<0.0001)among the treated groups and the control group, the treated ones inhibited the expression of the gene; the level of the CSN3gene expression between the treated groups was no significant difference, but it was very significant difference with the control group respectively(p<0.0001), treated ones almost completely inhibited the expression of the gene; the level of the CSN1S1gene expression showed significant difference highly among treated and control groups(p<0.0001), the treated groups inhibited the expression of the gene; the difference of the CSN2expression was significant among the three groups(p<0.0001), the treated ones inhibited the CSN2expression. Treated groups inhibited the synthesis of a-casein and β-casein, and it was not different significant from the control group.Phe-phe on milk protein synthesis:C promoted the CSN1S2expression, D inhibited the expression of the gene, there were not significant differences among the treated ones and the control one; the LGB expression of the treated groups was suppressed, and the difference was not significant between them, but respectively, it was significant different with the control group (p<0.05); the treated groups promoted the CSNS expression, the difference was not significant among the treated groups and the control group; D promoted CSN1S1expression, C inhibited the expression of the gene, there was significant difference among treated groups and the control one(p<0.05).the treated groups inhibited the synthesis of α-casein and β-casein, and it was not different significant from the control group.Met-lys on milk protein synthesis:E promoted the CSN1S2expression, F inhibited the gene expression, the difference was not significant among the groups; the LGB expression of the treated groups was inhibited, and the difference was not significant, but it was very significant compared with the control group respectively(P<0.0001).The treated ones inhibited the CSN3expression, F was different significantly from E and the control group (P<0.0001),the CSN1S1expression of the treated groups was inhibited, and the difference was significant among the three groups (P<0.0001); the treated groups inhibited the CSN2expression, and the difference was not significant between them, but it was different significantly compared respectively with the control group (P<0.0001), F promoted the expression of alpha-casein in the mammary gland cells, and inhibited the synthesis of β-casein, E inhibited the synthesis of casein, there was not significant difference among the treated ones and the control one. |
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