Responses Of Arginine To Milk Casein Synthesis And Its Manipulating Mechanism In Mammary Epithelial Cells From Lactating Cow

In this experiment, the mammary epithelial cell of Holstein lactating cow (CCMEC) was used for the model to assay the effects of different Arg concentrations on casein synthesis and its mechanism in vitro. The study was conduct From the level of cell proliferation、mRNA Transcription of casein gene and signal transduction related with casein synthesis genes. The purpose is to obtained a general regularity of which Arg can affect casein synthesis screening the best Arg concentration when casein synthesis was highest, reveal the cellular and molecular mechanisms,and provide a theoretical basis for improving milk protein and quality.The study included three parts.Exp.1Culture of mammary epithelial cells in vitroThe mammary epithelial cells were obtained with collagenase digestion method from healthy Holstein lactating cow and purified by trypsin. Pure epithelial cells were performed by passages culture、cell cryopreservation and recovery. the abtained cells were identificated by morphology, growth characteristics, immunohistochemistry, and biological functions. The cell growth curve was made by Trypan blue counting method and Expression of CK-18was identificated by immunohistochemical method. RT-PCR technique was used for detecting gene expression of CSN1S1、CSN1S2、CSN2、CSN3. Most of the isolated and resuscitated cells were exhibited a cobble-stone-like shape, connected tightly, with a clear boundary, and extended monolayer. the growth curve conformed to the rule of "S" sigmoid curve for proliferating cells with a lag-phase, exponential phase, and steady phas. The time for cell went into exponential was at the3rd day and steady phas at the7th day. CK-18immunohistochemical identification results showed a strong positive,IT confirmed that the cells we obtained were typical epithelial cells. RT-PCR results show that the CSN1S1, CSN1S2, CSN2, and CSN3gene expressed effectively in the cell,this confirmed these cells have the specific biological function of mammary epithelial cells.Exp.2Responses of Arginine levels to casein synthesis in cattle mammary epithelial cellIn this experiment, the Chinese Holstein cattle mammary epithelial cell(CCMEC) was used as the model to assay the effects of different Arg concentrations on α-、β-casein synthesis. The experiment was conducted with a completely randomized1×7factorial. Arg0-time group(0mg/L)was set as control,and Arg0.25-time group (69.50mg/L),0.5-time group (139mg/L),1-time group (278mg/L),2-time group (556.00mg/L),4-time group (1112.00mg/L),8-time group (2224mg/L) as treat group. α-、β-casein synthesis were tested by the ELISA method. The result showed that:compared with the control group, the treat group except for the Arg0.25-time and Arg8-times group was significantly different on a s-casein (P<0.05), a s-casein synthesis was the highest in Arg2-times group. On β-casein,the law was the same except for the Arg0.25time group.Exp.3Responses of Arginine levels to growth in cattle mammary epithelial cellIn this experiment, the Chinese Holstein cattle mammary epithelial cell(CCMEC) was used as the model to assay the effects of different Arg concentrations on cell growth and proliferation. The experiment was designed as the same as Exp.2. The method of trypan blue staining was used to draw the cell growth curves. The proliferative activity of cells were detected by MTT method at24h,48h and72h, respectively. The results show that, cell growth curves fit to a "S" shape in all the group. At the3rd day the cells got into exponential phases and then showed stationary phases at the7th day. The number of cells in Arg2-times group were higher than other groups. Compared with the control group, the promotion efficiency of Arg0.25-time to4-time groups were significantly high (P<0.05); It was however that, there were significant differences in the promotion efficiency of all the groups at both48h (P<0.05) and72h(P<0.05). Exp.4Effects of Arginine levels on the expression of casein gene and signal transduction genes that related with casein synthesis on cattle mammary epithelial cellIn this experiment, the Chinese Holstein cattle mammary epithelial cell(CCMEC) was used as the model to assay the effects of different Arg concentrations on cell growth and proliferation. The experiment was designed as the same as Exp.2. Real-Time PCR was applied to detect relative expression levels of CSN1S1, CSN1S2, CSN2, CSN3, JAK2, STAT5, mTOR, S6K and4EBP1gene. The results showed that, except for the Arg0.25-time group and Arg8-times group, the gene expression of CSN1S2, CSN2, JAK2in other groups were significantly higher than the control group(P<0.05). Except for the Arg8-times group the gene expression of STAT5and mTOR in treat group were significantly higher than the control group(P<0.05). Except for the Arg0.5-time group the gene expression of other groups were higher for S6K but significantly lower for4EBP1than the control group (P<0.05). The expression of CSN1S1, CSN1S2, CSN2, CSN3, JAK2, STAT5, mTOR, S6K gene were highest in the Arg2-times group,it was however that the expression for4EBP1gene was the lowest. In conclusion, Arg play an important role in regulation of CSN1S1, CSN1S2, CSN2, CSN3gene transcription. The expression of CSN1S1, CSN1S2, CSN2, CSN3can be promoted by arginine at appropriate concentration and they were highest in the Arg2-times group which contained556mg/L arginine in the medium. Transcription and translation of casein gene can be regulated and controlled by Arg through activate the JAK2/STAT5and mTOR pathway. The expression of JAK2, STAT5, mTOR, S6K gene were highest and4EBP1gene was lowest in the Arg2-times group.