| With the deep understanding of the food safety, the stand or fall of milk quality and the level of milk production drew increasing concerns from the public. Researches of lactation related to signaling pathways and exploretion on important factors of lactation will contribute to providing the new experimental evidence and methods of the bovine milk quality and the level of milk output.Glycyl t RNA synthetase(GlyRS) is an important family member of aminoacyl-t RNA synthetase(aaRSs). Recent researches have found that aa RSs has a function of signals controlling apart from various classic interpretation. However, the expressions of GlyRS used in the process of mammogenesis development and studies on its modification have not yet been reported. On the basis of the early stage of the laboratory, experiment reseachers have found that GlyRS could be linked to the synthesis of milk protein. In this study, we aimed to analyze the effect of GlyRS on bovine mammary epithelia cells(BMECs), milk protein, milk lipid and milk lactose, and cell proliferation study. Consequently, to reveal initially the fact that GlyRS is a new key signal molecule that regulates the lactogenesis in BMECs.The research cultures in vitro to gain the purified BMECs through the tissue culture method.The purity and function of milk protein synthesis of cells were identified through description of tests on all substance in keratin 18(CK18) and β-Casein(CSN2) with the assistance of immunofluorescence(IF) and western blotting(WB).The model in cell culture in virto was established by adding 0.6mmol/L methionine(Met) or10% fetal bovine serum(FBS) in the culture solution. WB, IF and quantitative real-time PCR(q RT-PCR), posterion to addition of Met or FBS, were used to detect the secretion of lactose and triglyceride(TG), the number of DNA, the expressions of CSN2, GlyRS and p-GlyRS(T544,S704), and the nuclear localization of p-Gly RS(T544, S704). The results showed that the secretion of lactose and TG had been promoted, as well as the increasing success in the synthesis of DNA and the expressions of CSN2, GlyRS and p-GlyRS(T544, S704) were significantly increased(p<0.05) and the nuclear localization of p-GlyRS(T544, S704) was dramatically risen with the stimulus of Met or FBS. It suggests that Met or FBS can regulate the process of BMECs lactogenesis and cell proliferation by GlyRS.Using the purified BMECs respectively establish GlyRS overexpression(pGCMV-IRES-EGFP-GlyRS) model and a GlyRS model target against the shRNA. The secretion volume of lactose and TG, the number of DNA, the distribution of the cell cycle and the expression of GlyRS,p-GlyRS(T544), p-GlyRS(S704), mTOR, p-mTOR, S6K1, p-S6K1, SREBP-1, CSN2, Cyclin D1 and β-1,4-galactosyltransferase2(β4Gal-T2) were detected by WB, IF, qRT-PCR and flow cytometry(FCM). The results indicated that the secretion of lactose and TG, the DNA synthesis of BMECs, the numbers of cells in S and G2/M phase were all significantly increased in the condition of the overexpression of GlyRS(p<0.05). The protein expression levels of GlyRS, p-GlyRS(T544), p-GlyRS(S704), p-mTOR, p-S6K1, SREBP-1, Cyclin D1 and CSN2, and the mRNA expressions of β4Gal-T2 were significantly increased and decreased(p<0.05). Whereas, the protein all expression levels of mTOR and S6K1 showed no obvious change. It suggests that GlyRS could promote the synthesis of milk protein, milk fat and lactose by upgrading the expression levels of p-GlyRS(T544), p-GlyRS(S704), p-mTOR, p-S6K1, SREBP-1, β4Gal-T2 and Cyclin D1, meanwhile improve the ability of BMECs proliferation.In summary, GlyRS was an important signal molecule; it can take in the signal of the nutrient substance(such as Met and FBS). The possible modifying is as the following: when the GlyRS was stimulated with Met and FBS, its phosphorylation and nuclear localization were increase, the lactogenesis and cells proliferation would be enhance via upregulating the expressions of p-mTOR,p-S6K1, SREBP-1, β4Gal-T2 and Cyclin D1. |
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