干扰hENT1表达增加5-FU在胰腺癌SW1990细胞内浓度

Interfering hENT_1 expression raising 5-FU concentration in pancreatic cancer SW1990 cells

目的明确hENT1在5-FU跨膜转运和返流中的作用,以及返流对5-FU在细胞内浓度的影响。方法将能特异性下调hENT1的重组及对照质粒用脂质体法转染到SW1990细胞内,RT-PCR检测hENT1 mRNA表达情况。分别培养SW1990、pSilence-hENT1Si SW1990、pSilence-Control SW1990细胞,3组细胞均置于含5-FU100mg·L-1的DMEM中温浴。各组取等量细胞刮取液两份,一份用毛细管区带电泳测定5-FU含量、另一份作平行样本测定蛋白含量,最后计算细胞内5-FU浓度。结果pSilence-hENT1Si SW1990细胞hENT1 mRNA表达水平下调0.5。SW1990组用5-FU温浴后,细胞内5-FU浓度在早期迅速升高,120min后处于平台期。pSilence-hENT1Control-SW1990组与SW1990组相比无差异(P>0.05)。而pSilence-hENT1SiSW1990细胞内5-FU浓度早期上升较慢,但随着时间延长,浓度逐渐升高,120min后细胞内浓度稳定,高于对照组中浓度(P<0.05)。结论干扰hENT1表达可以提高5-FU在SW1990细胞内浓度。hENT1是SW1990细胞向外返流5-FU的主要通道。

Aim To investigate the effect of hENT1 on trans-member transport and back-flow of 5-FU in SW1990 cells, and the effect of back-flow of 5-FU in the concentration of 5-FU in SW1990 cells. Methods pSilence- hENT1 plasmid down-regulated hENT1 specifically and its control plasmid was separately transfected into SW1990 cells by liposome, hENTI mRNA expressions in cells were checked by RT-PCR. SW1990, pSilenee-hENT1 SiSW1990,pSilence-Control-SW1990 cells were harvested alter incubation with DMEM containing 5-FU（ 100 mg· L^-1 ）. Then,two cell suspensions in each ceils were spliced,one for 5-FU detection and another for protein contents. Finally, the 5-FU concentration in a single cell was counted. Results The hENT1 mRNA expression in pSilenee-hENT1 Si-SW1990 cells was down-regulated to 0.5 compared with that of controls. After 5-FU ineubation,the concentration of 5-FU in SW1990 cells was raised rapidly early, then, a concentration plateau was achieved after 120 min. There was no difference of concentration between SW1990 cells and pSilence-hENT1 Control-SW1990 cells （P 〉 0.05）. In pSilence-hENT1 Si-SW1990 cells, in contrast, the concentration of 5-FU in cells was raised slowly early, the coneentration of 5-FU in pSilence- hENT1 Si SW1990 cells located at a curve plateau after 120 rain,much higher than that in SW1990 cells （P 〈 0. 05）. Conclusions Down-regulating hENT1 expression could raise 5-FU concentration in SW1990 cells. hENT1 might be the main channel for 5-FU back-flow out of cell in SW1990.