【摘要】 以体外培养的奶牛乳腺上皮细胞为模型,研究了RagD对细胞中β-酪蛋白(CSN2)的合成及蛋氨酸调节CSN2合成信号通路的影响。采用实时荧光定量PCR(q RT-PCR)和蛋白质免疫印迹(WB)检测添加蛋氨酸后细胞中CSN2与Rag D的表达;对Rag D进行过表达和抑制,用q RT-PCR和WB检测细胞中CSN2、mTOR和p-mTOR的表达。结果表明:添加蛋氨酸使细胞中CSN2和RagD的表达显著增加;RagD过表达时,细胞中CSN2与p-mTOR的表达显著增加,mTOR表达变化不显著,此时添加蛋氨酸,细胞中CSN2与p-m TOR的表达继续显著增加;RagD抑制时,细胞中CSN2与p-m TOR的表达显著下降,mTOR表达变化不显著(P>0.05),此时添加蛋氨酸,不能使细胞中CSN2与p-m TOR的表达恢复。RagD作为信号分子,能接收并传递蛋氨酸信号,使细胞中mTOR磷酸化,正向调节奶牛乳腺上皮细胞中CSN2的表达。更多还原

【Abstract】 The experiment was conducted to investigate the effects of Rag D on milk protein synthesis and the signaling pathway of methionine regulate β-casein(CSN2) synthesis in bovine mammary epithelial cells cultured in vitro. The expression of CSN2 and Rag D were detected by real-time quantitative PCR(q RT-PCR) and western blotting(WB) in cells treated with methionine. The expression of CSN2,m TOR and p-m TOR were detected by q RT-PCR and WB in cells after Rag D overexpression and inhibition. The results showed as follows: the expression of CSN2 and Rag D were significant increase after treated with methionine.The expression of CSN2 and p-m TOR were significant increases and significant decreases, after Rag D overexpression and inhibition, but the expression of m TOR was no significant change. In Rag D overexpression group, the expression of CSN2 and p-m TOR were significant increases(P<0.01) after methioninethe was added, but in Rag D inhibition group, the expression of CSN2 and p-m TOR were no increases after methioninethe was added. The study suggests that as signaling molecules, Rag D can receive and pass methionine signal, phosphorylation the m TOR, and positively regulate the expression of CSN2 in bovine mammary epithelial cells.更多还原