14-3-3β对奶牛乳腺上皮细胞内质网应激相关基因以及泌乳效应的影响

Effect of 14-3-3β on Gene Expressions of the ER Stress and Lactation in BMECs

以奶牛乳腺组织和正常生长的奶牛乳腺上皮细胞为研究对象,通过使用qRT-PCR检测青春期、泌乳期和退化期奶牛乳腺组织中14-3-3β(tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein beta)、eIF2α(eukaryotic translation initiation factor 2 subunit alpha)和ATF4(activating transcription factor 4)的mRNA表达水平以及Western Blot检测14-3-3β、p-eIF2α和ATF4的蛋白表达水平。结果表明,在泌乳期的奶牛乳腺组织中14-3-3β蛋白表达水平最低,而p-eIF2α蛋白表达水平最高。运用RNAi技术将乳腺上皮细胞中14-3-3β基因敲除,用毒胡萝卜素(TG,Thapsigargin)诱导细胞内质网应激,检测14-3-3β、eIF2α、ATF4、CHOP(DNA damage inducible transcript 3)、Caspase-3的mRNA表达水平的变化,同时检测β-酪蛋白的mRNA和蛋白水平的表达变化,以及用甘油三酯检测试剂盒和MTT细胞增殖检测试剂盒检测奶牛乳腺上皮细胞的乳脂含量、增殖情况的变化。结果表明,在14-3-3β敲除之后,eIF2α、ATF4、CHOP和Caspase-3的mRNA表达水平显著升高,而β-casein的mRNA和蛋白表达水平都显著降低,同时乳脂含量和细胞增殖率也显著下降。揭示14-3-3β参与奶牛乳腺上皮细胞内质网应激关键因子eIF2α和ATF4的调控并调节泌乳效应,进一步完善了14-3-3β与泌乳调控和内质网应激有关的分子机制。

In this study, growing normally bovine mammary epithelial cells(BMECs)and mammary issues were used as experimental subjects. The mRNA expressions of 14-3-3β, eIF2αand ATF4 in mammary glands during puberty, lactation and degeneration were detected by qRT-PCR and the protein expressions of 14-3-3β, p-eIF2αand ATF4 were detected by Western Blot. These results indicated that the expression level of 14-3-3βprotein was the lowest in mammary gland of lactation, while the expression level of p-IF2αprotein was the highest. RNAi strategy was used to knockdown 14-3-3βin BMECs. Using TG to induce the ER stress and detect the mRNA expression levels of 14-3-3β, eIF2α, ATF4, CHOP, Caspase-3, at the same time, the expression levels ofβ-casein mRNA and protein were detected,and the changes of milk fat content and proliferation and cell viability of BMECs were detected by Triglycerides assay kit and MTT cell proliferation assay kit. The results showed that mRNA expression levels of eIF2α, ATF4, CHOP and Caspase-3 were significantly increased but the mRNA and protein expression levels ofβ-casein significantly decreased, and milk fat content and cell proliferation rate also decreased significantly after 14-3-3βknockdown. This study revealed that 14-3-3βis involved in the regulation of ATF4 and eIF2α, a key factor of ER stress and regulates effect of lactation in BMECs, further improved the molecular mechanism of 14-3-3βrelated to ER stress and regulation of lactation.