脂多糖对奶牛乳腺上皮细胞体外生长及细胞内固醇调节元件结合蛋白-1、肝脏X受体基因mRNA转录水平的时序性影响

Sequential effect of lipopolysaccharide on growth in vitro of bovine mammary epithelial cells and transcription levels of sterol regulatory element binding protein 1 and liver X receptor mRNAs

目的分析脂多糖(lipopolysaccharide,LPS)对奶牛乳腺上皮细胞(bovine mammary epithelial cells,BMECs)体外生长及细胞内固醇调节元件结合蛋白-1(sterol regulatory elementbinding protein 1,SREBP-1)和肝脏X受体(liver X receptor,LXR)基因mRNA转录水平的时序性影响。方法原代培养BMECs,用LPS(终浓度为10μg/m L)分别刺激BMECs 0、1、3、6、12、24和48 h后,显微镜下观察细胞状态,采用实时荧光定量PCR(q RT-PCR)法检测BMECs中SREBP-1及LXR基因mRNA的时序性表达水平。结果 LPS刺激BMECs 0、1、3 h后的生长状态良好,细胞形态饱满,胞浆丰富,无任何肉眼可见变化;LPS刺激6 h后,BMECs开始出现明显收缩、变圆,细胞间隙增大,细胞突起减少。与LPS刺激0 h比较,LPS刺激BMECs 1 h后可显著下调LXR基因mRNA转录水平(P<0.05),刺激3 h后可显著下调SREBP-1基因mRNA转录水平(P<0.05),且均随时间的延长呈逐渐降低趋势(P<0.05),均于LPS刺激12 h时达最低,刺激24及48 h后LXR及SREBP-1基因mRNA转录水平均明显高于12 h,24和48 h间差异无统计学意义(P>0.05)。结论 LPS可对BMECs产生严重损伤,能有效抑制BMECs中SREBP-1及LXR基因mRNA转录水平,且具有一定的时间依赖性。

Objective To investigate the sequential effect of lipopolysaccharide（LPS） on the growth in vitro of bovine mammary epithelial cells（BMECs） and the m RNA transcription levels of sterol regulatory element binding protein 1（SREBP-1） and liver X receptor（LXR）. Methods Primary culture of BMECs were stimulated with LPS at a final concentration of 10 μg/m L for 0, 1, 3, 6, 12, 24 and 48 h, observed for morphology by microscopy, and determined for sequential expression levels of SREBP-1 and LXR by real-time fluorescent quantitative PCR（q RT-PCR）. Results The BMECs 0, 1 and 3 h after stimulation with LPS grew well, of which the shape was plump with plenty cytoplasm and without any visual change. However, after stimulation with LPS for 6 h, BMECs were obviously shrunk and round, between which the gap increased, and cell processes decreased. The transcription level of LXR m RNA in BMECs 1 h and that of SREBP-1 m RNA 3 h after stimulation with LPS decreased significantly compared with that before stimulation（each P〈0. 05）, both of which showed decreasing tendencies as time went on and reached the minimum 12 h after stimulation. The transcription levels of LXR and SREBP-1 m RNAs 24 and 48 h after stimulation showed no significant difference（P〈0. 05）, which were significantly higher than those 12 h after stimulation. Conclusion LPS may cause the severe damage of BMECs and effectively inhibit the transcription levels of SREBP-1 and LXR m RNAs in BMECs in a certain timedependent pattern.