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目的建立Ezrin基因表达缺失的Hep G2细胞株,并研究其在胆汁淤积时对Hep G2细胞胆汁酸转运蛋白多药耐药相关蛋白2(multidrug resistance-associated protein 2,MRP2)的影响。方法不同位点干扰的Ezrin-shRNA质粒转染Hep G2细胞,筛选Ezrin基因表达缺失的Hep G2细胞。分别提取细胞总蛋白和膜蛋白,检测Ezrin基因表达缺失是否影响Hep G2细胞胆汁酸转运蛋白MRP2的表达。结果 RT-q PCR结果证实,Ezrin-shRNA#3质粒转染Hep G2细胞后成功抑制了Ezrin基因表达,抑制率为70%。蛋白免疫印迹表明,Ezrin基因表达缺失后,Hep G2细胞胆汁酸转运蛋白MRP2表达增高,但与对照组相比,差异无统计学意义(P>0.05)。蛋白免疫共沉淀检测生物素化MRP2膜蛋白表明,Ezrin基因表达缺失后,无论是胞浆中还是细胞膜上,MRP2膜蛋白均表达增高(P<0.05)。结论 Ezrin基因表达缺失后能够上调Hep G2细胞中MRP2膜蛋白,使其表达增高。
Objective To establish Hep G2 cell line with deletion of Ezrin gene expression and study its effect on bile acid transporter multidrug resistance-associated protein 2 (MRP2) in Hep G2 cells during cholestasis. Methods Hep G2 cells were transfected with Ezrin-shRNA plasmid with different sites and Hep G2 cells with Ezrin gene deletion were screened. The total cellular protein and membrane protein were extracted respectively to detect whether the expression of Ezrin gene affected the expression of bile acid transporter (MRP2) in Hep G2 cells. Results The results of RT-q PCR confirmed that the Ezrin gene was successfully inhibited in Ezrin-shRNA # 3 plasmid transfected Hep G2 cells with the inhibition rate of 70%. Western blotting showed that the expression of MRP2 in Hep G2 cells increased after Ezrin gene deletion, but there was no significant difference compared with control group (P> 0.05). Protein co-immunoprecipitation assay of biotinylated MRP2 membrane protein showed that the expression of MRP2 protein in both cytoplasm and plasma membrane was increased (P <0.05) after Ezrin gene expression was absent. Conclusion The deletion of Ezrin gene can up-regulate the expression of MRP2 membrane protein in Hep G2 cells.