Objective To prepare retinol binding protein 4( RBP4 ) interfering vector and observe its impact on ERK1/2 signaling pathway in human liver LO 2 cell line . Methods Two pSilencer?4.1- RBP 4 vectors targetingrnRBP4 expressing frame 5 'or 3 'terminal region and the eukaryotic expression vector of pSecTag-RBP4 were constructed and identified by sequencing and enzyme cutting, then transfected into LO2 cells, whose interfering efficiency were evaluated by RT-PCR and Western blot, and pSilencer?4. l-RBP4-5'was used for follow-up experiment. The protein expression and phosphorylation of ERK1/2 were evaluated by Western blot, the content of glucose remained in LO2 cells supernatant was measured by the method of glucose oxidase peroxides( GOD-POD ). Results The interfering vector and eukaryotic expression vector were successfully prepared and RBP4 expression in LO2 was effectively inhibited. The protein expression of ERK1/2 did not significantly decrease compared with that of the blank control group, but the phosphorylation level of ERK1/2 in pSecTag-RBP4 group significantly decreased, while significantly increased in pSilencer 4. 1-RBP4-5 group. The glucose intake by insulin stimulation significantly decreased in pSecTag-RBP4 group, while the glucose uptake markedly increased in pSilencer 4. 1-RBP4 group. Conclusion The interfering vector and eukaryotic expression vector are successfully prepared and used to study phosphorylation of ERK1/2 and glucose uptake in LO2 cells.%目的 制备人视黄醇结合蛋白4(RBP4)分子RNAi载体,检测其干涉人肝细胞系LO2中RBP4的表达后对ERK1/2表达及磷酸化和葡萄糖摄取的影响.方法 分别构建针对RBP4基因表达框5′端和3′端的干涉载体及真核表达载体,经测序和酶切鉴定后将载体转染LO2,RT-PCR和Western blot法确定其干涉RBP4表达的效率,选择干涉较高的载体用于后续实验;Western blot检测ERK1/2的表达及磷酸化水平的改变,葡萄糖氧化酶法检测上清液中葡萄糖的残留量.结果 构建的真核表达载体和干涉载体经酶切、测序鉴定结果正确,并有明显的高表达或干涉效果.Western blot结果表明ERK1/2的表达量无明显变化,RBP4高表达组其磷酸化显著下降,RBP4干涉组其磷酸化显著上升.胰岛素刺激后RBP4高表达组的葡萄糖摄取明显下降,RBP4干涉组的葡萄糖摄取明显增加.结论 成功制备RBP4基因的干涉载体并应用于LO2细胞中ERK1/2通路的研究,为后续RBP4的进一步功能研究奠定基础.
展开▼
