siRNA干扰Caco-2细胞鞘磷脂合酶2 (SMS2)基因对药物转运体的影响

 目的  研究鞘磷脂合酶2 (sphingomyelin synthase 2,SMS2)缺损对人结肠癌细胞(colon cancer cell,Caco-2)中药物转运体P 糖蛋白(P-glycoprotein,P-gp)及多药耐药相关蛋白2 (multidrug resistance-associated protein 2,MRP2)的表达与功能的影响。方法  SMS2特异性siRNA转染至Caco-2细胞。采用RT-PCR和real-time PCR检测SMS2-特异性siRNA的干扰效率;采用Western blot法检测siRNA转染后P-gp和 MRP2蛋白表达的变化;采用胞内蓄积试验,通过HPLC检测P-gp及MRP2专一性底物罗丹明123及普伐他汀的蓄积含量,分析下调SMS2水平对Caco-2细胞中P-gp及MRP2功能的影响。结果  应用siRNA下调SMS2水平后,P-gp和MRP2的蛋白质水平均显著下调;胞内蓄积实验结果显示罗丹明123及普伐他汀的蓄积量明显增加,说明P-gp和MRP2的外排功能减弱。结论  SMS2基因表达下调对Caco-2细胞中P-gp和MRP2的表达以及功能均有显著影响。

Effects of sphingomyelin synthase 2 (SMS2) interefered by siRNA on drug transporters in Caco-2 cells

Objective  To study the effect of sphingomyelin synthase 2 (SMS2) defect on the expression and function of drug transporters P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) in human colon cancer (Caco-2) cells.Methods  SMS2-specific siRNA was transfected into Caco-2 cell.RT-PCR and real-time PCR were employed to detect the efficiency of siRNA.Western blot was used to examine the protein expressions of P-gp and MRP2.Intracellular accumulation experiment was carried out to evaluate the effect of SMS2 down-regulation level on the functions of P-gp and MRP2 in Caco-2 cells.HPLC analysis was used to decect the accumulation amounts of parvastain and rhodamine 123,the specific substrates of P-gp and MRP2.Results  The expressions of P-gp and MRP2 were down regulated in SMS2 knockdown cells treated by siRNA.Cellular accumulation experiments revealed that the excretion function of P-gp and MRP2 in SMS2 deficient cells was decreased,as the accumulation of rhodamine 123 and parvastain was increased significantly.Conclusions  The down-regulation of SMS2 gene on the expression and function of P-gp,MRP2 in Caco-2 cell.