RBP4分子RNAi载体的制备及对人肝细胞系LO2中ERK信号通路的影响

Preparation of RBP4 interfering vector and its impact on ERK signaling pathway in human liver LO2 cell line

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。

Objective To prepare retinol binding protein 4（RBP4） interfering vector and observe its impact on ERK1/2 signaling pathway in human liver LO2 cell line.Methods Two pSilencerTM 4.1-RBP4 vectors targetingRBP4 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 pSilencerTM 4.1-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 pSilencerTM 4.1-RBP4-5 group.The glucose intake by insulin stimulation significantly decreased in pSecTag-RBP4 group,while the glucose uptake markedly increased in pSilencerTM 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.