【摘要】 利用脂多糖（LPS）诱导奶牛乳腺上皮细胞（Bovine mammary epithelial cells,BMECs）建立炎症细胞模型，采用RT-qPCR检测BMECs中炎症因子及AKT1 mRNA表达变化；采用BSP(Bisulfite sequencing PCR)检测AKT1基因启动子区域甲基化水平；采用组织细胞甘油三酯（Triglyceride,TG）含量酶法测定试剂盒检测细胞中甘油三酯含量；采用Bovine lactose ELISA KIT检测细胞中乳糖含量；采用Western blot技术检测β-casein、AKT1、PAKT1、DNA甲基转移酶DNMT1和DNMT3A蛋白表达情况。试验结果表明，LPS诱导后奶牛乳腺上皮细胞中甘油三酯、乳糖和β-酪蛋白合成减少，AKT1表达量减少，P-AKT1蛋白表达量也减少，同时DNMT1和DNMT3A蛋白表达量增加，AKT1基因启动子甲基化水平升高，表明DNA甲基化参与乳腺炎条件下奶牛泌乳调控，LPS也通过促进DNA甲基转移酶表达提高AKT1基因甲基化水平而抑制AKT1表达，抑制乳脂、乳糖和乳蛋白合成。更多还原

【Abstract】 In this study, lipopolysaccharide(LPS) was used to induce bovine mammary epithelial cells(Bovine mammary epithelial cells, BMECs) to establish a cellular inflammatory model. The expression of inflammatory factors and AKT1 mRNA in BMECs were detected by RT-qPCR; the methylation level of AKT1 gene promoter region was detected by BSP(Bisulfite sequencing PCR); the content of triglyceride(TG) in cells was detected by the enzyme assay kit; Bovine lactose ELISA KIT was used to detect the lactose content in cells. The protein expressions of β-casein, AKT1, P-AKT1,DNA methyltransferase DNMT1 and DNMT3A were detected by Western blot. The results showed that the synthesis of triglyceride, lactose and β-casein protein in the mammary epithelial cells of dairy cows induced by LPS all decreased, the expression of AKT1 decreased, the expression of P-AKT1 protein decreased, the protein expressions of DNMT1 and DNMT3A increased, and the methylation level of AKT1 gene promoter increased, indicating that DNA methylation was involved in the regulation of lactation of dairy cows under the condition of mastitis, and LPS could also increase the methylation level of AKT1 gene by promoting the expression of DNA methyltransferase, thus inhibiting the expression of AKT1, thereby inhibiting the synthesis of triglyceride, lactose and casein.更多还原