蛋氨酸二肽对Transwell小室培养的奶牛乳腺上皮细胞中乳蛋白合成的影响

Effects of Methionyl⁃Methionine on Milk Protein Synthesis in Transwell Chamber Cultured Bovine Mammary Epithelial Cells

为明确奶牛乳腺中小肽的吸收和利用机制,本试验研究了蛋氨酸二肽(Met-Met)在Transwell小室培养的奶牛乳腺上皮细胞(BMECs)中的摄取及其对乳蛋白合成的影响。首先将BMECs培养在Transwell小室中,待BMECs形成细胞层后,分别在Transwell上室和下室培养液中添加0.5 mmol/L的Met-Met,以不添加Met-Met作为对照。培养24 h后,收集BMECs,采用Western blot检测β-酪蛋白(β-casein)和小肽转运载体2(PepT2)蛋白的相对表达量以及哺乳动物雷帕霉素靶蛋白(mTOR)、核糖体蛋白S6激酶1(S6K1)和蛋白激酶B(AKT)的磷酸化水平。结果显示:BMECs在Transwell小室中培养6 d后,细胞层跨膜电阻达到平台期,反映细胞层紧密性良好;Transwell上室和下室培养液中添加Met-Met显著增加了BMECs中β-casein和PepT2的蛋白相对表达量(P<0.05);Transwell下室培养液中添加Met-Met显著提高了mTOR、S6K1和AKT蛋白的磷酸化水平(P<0.05)。以上结果表明,Transwell上室和下室培养液中的Met-Met均可被BMECs摄取用于乳蛋白的合成,PepT2在BMECs基底膜小肽吸收和顶膜小肽重吸收过程中发挥重要作用,基底膜Met-Met通过激活mTOR和AKT信号通路促进乳蛋白的合成。

To investigate the transport and utilization mechanism of small peptides in bovine mammary epitheli-al cells(BMECs),the uptake and its effect on milk protein synthesis of methionyl-methionine(Met-Met)in Transwell chamber cultured BMECs was studied in this experiment.BMECs were firstly cultured in Transwell chamber,and the 0.5 mmol/L Met-Met was added into the upper and lower chamber medium of Transwell af-ter cell layer formed,respectively.The medium without Met-Met was used as control.The BMECs were col-lected after 24 hours of culture.Western blot was used to detect the protein relative expression levels ofβ-case-in and oligopeptide transporter 2(PepT2),and the phosphorylation levels of mammalian target of rapamycin(mTOR),ribosomal protein S6 kinase 1(S6K1)and protein kinase B(AKT).The results showed that,after 6 days culture,the trans-epithelial electrical resistance of the BMECs layer reached the plateau stage,in-dicating good cell layer tightness;the addition of Met-Met in both upper and lower chambers medium of Tran-swell significantly increased the protein relative expression levels ofβ-casein and PepT2;the addition of Met-Met in the Transwell lower chamber medium significantly improved the phosphorylation levels of mTOR,S6K1 and AKT.These results indicate that the small peptides in both upper and lower chambers medium of Tr-answell can be absorbed and used for milk protein synthesis by BMECs,PepT2 plays an important role in the absorption of small peptides in basement membrane and the reabsorption of small peptides in apical membrane of BMECs,and Met-Met in basement membrane promotes milk protein synthesis by activating mTOR and AKT signaling pathways.