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目的研究胆汁淤积时Sp1对MRP3、MRP4表达的影响,以完善MRP3、MRP4表达调控机制。方法构建Sp1过表达质粒及Sp1 siRNA片段,分别转染HepG2细胞,筛选稳转细胞株。提取细胞总RNA和总蛋白,RT-qPCR检测MRP3、MRP4 mRNA水平的变化,Western blot检测MRP3、MRP4蛋白水平的变化。结果成功构建了Sp1-OE-HepG2和Sp1siRNA-HepG2稳转细胞株。在Sp1-OE-HepG2细胞中,MRP3、MRP4 mRNA和蛋白水平表达均增高,其中MRP3 mRNA水平表达增高2.8倍,MRP3蛋白水平表达增高3.0倍;MRP4 mRNA和蛋白水平分别增高3.2倍和2.5倍;而在Sp1 siRNA-HepG2细胞中,MRP3、MRP4则表达下降,其中MRP3 mRNA表达降低52%,MRP4 mRNA表达降低58%,MRP3蛋白表达降低57%,MRP4蛋白表达降低60%。结论转录因子Sp1能够调控胆酸转运蛋白MRP3、MRP4的表达。
Objective To study the effect of Sp1 on the expression of MRP3 and MRP4 in cholestasis to improve the regulation of MRP3 and MRP4 expression. Methods Sp1 overexpression plasmid and Sp1 siRNA fragment were constructed and transfected into HepG2 cells respectively to screen stable cell lines. The total cellular RNA and total protein were extracted, the mRNA levels of MRP3 and MRP4 were detected by RT-qPCR, and the changes of MRP3 and MRP4 protein were detected by Western blot. Results Sp1-OE-HepG2 and Sp1 siRNA-HepG2 cells were successfully constructed. The mRNA and protein levels of MRP3 and MRP4 increased in Sp1-OE-HepG2 cells, including MRP3 mRNA level increased 2.8-fold and MRP3 protein level increased 3.0-fold; MRP4 mRNA and protein levels increased 3.2-fold and 2.5-fold respectively; In Sp1 siRNA-HepG2 cells, the expression of MRP3 and MRP4 decreased, MRP3 mRNA expression decreased by 52%, MRP4 mRNA expression decreased by 58%, MRP3 protein expression decreased by 57% and MRP4 protein expression decreased by 60%. Conclusion The transcription factor Sp1 can regulate the expression of MRP3 and MRP4.