葡萄糖对SVAREC细胞P-糖蛋白转运和表达的影响

The Investigation of the Effect of High Glucose on Substrate Transport and Expression of P-gp (Membrane Transporter) in SVAREC Cells

目的探讨葡萄糖对内皮细胞SVAREC膜转运蛋白P-糖蛋白的底物转运和表达的影响。方法 1.用含10％胎牛血清的DMEM培养液将SVAREC细胞传入24孔板,等细胞贴壁后将细胞分成4组,分别换以下条件刺激,A组:低葡萄糖(5.5mmoL/L);B组:高葡萄糖(25mmol/L);c组:低葡萄糖(5.5mmol/L)+Verapamil(10^(-4)Mol);D组:高葡萄糖(25mmol/L)+Verapamil(10^(-4)/Mol);每组都同时用不同浓度的秋水仙碱(0;12.5;25;50ug/ml)刺激72小时,以MTT法测定细胞的耐受性。2.按上述方法将细胞传入培养皿中,待细胞贴壁后,分别换葡萄糖浓度为5.5mmol/L,25mmol/L和50mmol/L的DMEM培养液继续培养72小时,提取细胞膜蛋白,Westem-blot检测P-糖蛋白的蛋白表达。结果高糖处理的细胞对秋水仙碱(P-糖蛋白的转运底物)细胞毒的耐受性明显低于低糖处理的细胞,在秋水仙碱浓度为25ug/ml时,差异最为显著(P<0.01),Verapamil(P-糖蛋白转运的特异抑制剂)共处理的低糖组细胞对秋水仙碱的耐受性显著降低(秋水仙碱浓度为25ug/L时,P<0.01),而Verapamil处理后的高糖和低糖组细胞对秋水仙碱的耐受性无显著性差异。Westem-blot结果显示5.5mmol/L葡萄糖刺激的细胞P-糖蛋白蛋白表达明显高于25mmoL/L和50mmoL/L葡萄糖刺激的细胞,而25mmol/L和50mmol/L之间没有显著差异。结论高糖能够降低内皮细胞P-糖蛋白对其底物的转运和表达,增加其底物秋水仙碱在细胞中的积累,使细胞的耐受性下降。

Objective To investigate the effect of glucose on P-glycoprotein protein expression and transport activity by researching the re- sistance of Endothelial cell line SVAREC cells to colchicine-the substrate of P-gp. Methods SVAREC cells were cultured and dividel into four groups, A: Low glucose (5.5mmol/L); B: high glucose (25mmol/L); C: low glucose (5.5mmol/L) with Verapamil(10^(-4)Mol); D: High glucose (25mmol/L) with Verapamil (10^(-4)Mol) .At same time evrery group was stimulated with Colchicine (0; 12.5; 25; 50ug/ml) in 72 hours. Then MTT metabolism was used to measure the resistance of cells. The protein expression of P- glycoprotein was decided by western -blot assay. Results The resistance of cells incubation with high glucose to Colchicine is significant lower than cells incubation witl low glu- case. Following stimulated with verapamil, the resistance of cells incubation with low glucose to Colchicine decreased significantly, but there isn' t difference significantly between high and low glucose stimulated with verapamil. The protein expression of P-glycoprotein is down- regulated by high glucose in SVAREC. Conclution High glucose decreased the protein expression and the transport of P- glycoprotein, increased the accumu- lation of its substrate, then deduced the resistance of cells.