染色质重建因子BRG1介导Ile调节牛乳腺上皮细胞乳合成分子机理研究

Study on molecular mechanism of chromatin remodeling factor BRG1 in the stimulation of Ile on milk synthesis in bovine mammary epithelial cells

试验旨在探究染色质重建因子BRG1(Brahma-related gene1)介导异亮氨酸(Isoleucine,Ile)在奶牛乳腺上皮细胞中影响乳合成分子机理。在细胞中添加不同浓度Ile,采用甘油三酯试剂盒、脂滴染色检测奶牛乳腺上皮细胞(Bovine mammary epithelial cells,BMECs)中乳脂肪含量变化,Western blot检测乳蛋白含量变化及乳合成相关信号通路分子(S6K1和SREBP-1c)表达水平变化;添加磷脂酰肌醇3激酶(PI3K)抑制剂LY294002与最适浓度Ile后,检测乳合成调节相关蛋白表达;检测添加最适浓度Ile同时敲低BRG1对乳蛋白、乳脂肪合成相关信号通路分子影响。结果表明,Ile以剂量依赖性方式促进BMECs乳合成。BRG1正向调控S6K1磷酸化和SREBP-1c表达。Ile显著上调BRG1蛋白水平,而抑制PI3K途径后BRG1蛋白水平显著下降,且Western blot结果显示添加Ile通过PI3K途径调控S6K1磷酸化和SREBP-1c蛋白表达。综上,染色质重建因子BRG1可介导Ile通过调控S6K1磷酸化和SREBP-1c表达,调节BMECs中乳蛋白和乳脂肪合成。

The aim of this study was to explore the effects of Ile on milk synthesis in bovine mammary epithelial cells(BMECs)and the mediation of chromatin remodeling factor Brahma-related gene 1(BRG1)on this regulation.In this experiment,BMECs were treated with different doses of Ile.Milk fat synthesis in BMECs after Ile treatment were measured,triglyceride kit and lipid droplet staining methods.Milk protein and the changes of milk synthesis-related signal pathways(S6K1 phosphorylation and SREBP-1c expression)in cells were also detected using Western blot.After adding LY294002(PI3K inhibitor)and the optimal concentration of Ile,S6K1 phosphorylation and SREBP-1c expression were detected.The effects of knock down of BRG1 wer determined in the presence of Ile on these signaling pathways.The results showed that Ile promoted milk protein and fat synthesis in BMECs in a dose-dependent manner.BRG1 was required for Ile to stimulate S6K1 phosphorylation and SREBP-1c expression.The protein level of BRG1 decreased significantly after inhibition of the PI3K pathway,and Western blot results also showed that PI3K was required for Ile to stimulate S6K1 phosphorylation and SREBP-1c expression.In summary,these above results revealed that BRG1 was a key mediator for Ile to stimulate milk protein and fat synthesis in BMECs via regulating S6K1 phosphorylation and SREBP-1c expression.