摘要
目的:明确下调hENT1后对胰腺癌细胞株Sw1990跨膜转运吉西他滨的影响。方法:将能特异性下调hENT1的pSilence-hENT14.1-CMV neo的重组质粒以及对照质粒转染至胰腺癌Sw1990细胞内。采用G418筛选阳性克隆细胞,RT-PCR检测hENT1mRNA表达情况。培养Sw1990细胞、pSilence-hENT1Si-Sw1990、pSilence-Control-Sw1990细胞,3组细胞培养基中吉西他滨质量浓度均为100μg/mL,温育0,15,30min,1,2,4h后收集细胞。取等量细胞悬液两份各100μL,一份作为检测样本用毛细管区带电泳测定吉西他滨总量,另一份用作平行样本测定总蛋白含量。根据蛋白-细胞数量标准曲线来确定平行样本中细胞数,再用血细胞分析仪确定细胞体积,最后,计算单细胞内药物总量及浓度。结果:pSilence-hENT1Si-Sw1990细胞的hENT1mRNA表达水平下调50。Sw1990组用含吉西他滨(100μg/mL)的DMEM培养基温育后,细胞内药物质量浓度在30min达(55.1±10.4)μg/mL,60min达(70.2±7.8)μg/mL,120min后处于平台期(90.1±6.0)μg/mL。相比之下,pSilence-hENT1Si-Sw1990组细胞内质量药物浓度30min达(41.3±4.9)μg/mL,60min达平台期(51.3±11.8)μg/mL,与对照组相比,细胞内浓度处于较低水平(P<0.05)。pSilence-hENT1control-Sw1990组与Sw1990组相比差异无统计学意义(P>0.05)。结论:下调hENT1可以抑制胰腺癌细胞株Sw1990对吉西他滨的摄取,hENT1是吉西他滨跨膜转运的重要通道。
Aim: To investigate the effect of hENT1 down-regulation on gemcitabine transport in pancreatic cancer cell line (Sw1990). Methods: pSilence-hENT1 4. 1-CMV neo plasmid to specifically down-regulated hENT1 and its control plasmid were separately transfected into Sw1990 cells. Then the transfected cells were cloned with G418, and their hENT1 mRNA expressions were checked by RT-PCR. Cells were harvested after 15 min, 30 min, 1 hr, 2 hr, and 4 hr incubation with medium containing gemcitabine ( 100 μg/mL). Then, two 100μL ceil suspensions in each cells were spliced, one for gemcitabine detection and another for protein quantitation. Gemcitabine contents in cells were detected by means of capillary zone electrophoresis. The cell number in 100 μL cell suspensions was defined with standard curve of protein content vs cell number. Average cell volume was defined with hematological analyzer. The gemcitabine content and concentration in a single cell were finally counted. Results: The hENT1 mRNA expression in pSilence-hENT1Si-Sw1990 ceils were down-regulated to 50% in comparison to the control group. After gemeitabine incubation ( 100 μg/mL), the concentration of gemcitabine in Sw1990 cells was (55.1±10.4) μg/mL at 30 min, (70.2±7.8) μg/mL at 60 min and (90. 1 +6.0)μg/mL at 120 min. After 120 rain, gemcitabine plateau approached. In pSilence-hENT1Si-Sw1990 cells, however, gemcitabine concentration was (41.3±4.9) μg/mL at 30 min, and (51.3 ± 11.8) μg/mL at 60 rain, much low- er than those in Sw1990 cells ( P 〈 0. 05). After that a plateau was achieved. No difference in the concentration versus time curves was found between Sw1990 cells and pSilence-hENTlcontrol.Sw1990 (P 〉 0.05). Conclusion: bENT1 down-regulating decreases gemcitabine up-take in pancreatic cancer cell line Sw1990. It is implicated that bENT1 might be the key channel for gemcitabine membrane transport.
出处
《中国药科大学学报》
CAS
CSCD
北大核心
2008年第5期457-462,共6页
Journal of China Pharmaceutical University
基金
东南大学913/事业基金资助项目(No.9290001327)~~
关键词
RNA干扰
核苷载体
吉西他滨
毛细管区带电泳
胰腺癌细胞株
RNAi
nucleoside transporter
gemcitabine
capillary zone electrophoresis
pancreatic cancer cell line