GCS基因与MDR1基因在肝癌细胞多药耐药中的相关性研究

The GCS gene and MDR1 gene in the multidrug resistance of hepatoma cells:a correlation study

目的观察葡萄糖神经酰胺合酶(GCS)基因高表达对肝癌细胞HepG2内多药耐药基因(MDR1)表达的影响,探讨GCS基因参与肝癌多药耐药的作用机制。方法脂质体法将GCS基因真核表达质粒pEGFP-GCS导入肝癌细胞株HepG2中,应用G418筛选培养,反转录聚合酶链反应(RT-PCR)检测细胞GCS mRNA和MDR1 mRNA的表达,蛋白印迹法检测细胞GCS蛋白和P-糖蛋白(P-gp)的表达,四甲基偶氮唑蓝(MTT)法检测基因转染前后阿霉素对HepG2细胞半数抑制浓度(IC50)的变化。结果转染后HepG2细胞内GCSmRNA和蛋白的表达水平显著提高(P<0.05),而MDR1mRNA及P-gp的表达水平亦显著提高(P<0.05);同时,MTT法检测结果显示GCS基因转染后HepG2细胞对阿霉素的耐药性显著提高(P<0.05),IC50从(6.2±0.4)μg/ml上升到(11.6±1.0)μg/ml。结论高表达GCS可以上调HepG2细胞中MDR1的表达,使HepG2细胞发生多药耐药。

Objective To observe the impact of expression of glucosylceramide synthase （GCS） gene on the expression of multidrug resistance 1 （MDR1） gene in HepG2, and to explore the mechanism of GCS gene involving MDR of hepatocarcinoma. Methods The GCS gene eukaryotic expression vector pEGFP-GCS was transiently transferred into HepG2 cells by the lipofectamine method. HepG2 cells were then screened and cultured using G418. Thereafter, the expressions of GCS mRNA and MDR1 mRNA were detected by RT-PCR, the expressions of GCS protein and P-gp by Western blotting, and changes of half inhibitory concentration （IC50） of HepG2 affected by adriamycin before and after transfection by MTT method. Results After transfection, the expressions of GCS mRNA and protein in HepG2 were significantly increased （P0.05） and the expressions of MDR1 mRNA and P-gp increased as well （P0.05）. Meanwhile, MTT method showed a significant increase in MDR of HepG2 to adriamycin after transfection of GCS gene （P0.05）. And the IC50 was increased from （6.2±0.4）μg/ml to （11.6±1.0） μg/ml. Conclusion The overexpression of GCS gene could up-regulate the expression of MDR1 gene in HepG2 so as to result in MDR of HepG2.