转染胸苷磷酸化酶基因对胃癌细胞株氟尿嘧啶类药敏感性的影响

Improved sensitivity of gastric carcinoma cells to fluorouracil-related drugs by transfection of thymidine phosphorylase gene

目的 观察胃癌细胞株中胸苷磷酸化酶（TP）表达水平与其对化疗药物5-氟尿嘧啶（5-FU）及其前体药去氧氟尿苷和卡倍他滨敏感性的关系.方法 利用Lipdectamin 2000将质粒pEGFP-N1及重组质粒pEGFP-N1-TP转染入胃癌细胞株AGS中,在倒置荧光显微镜下观察绿色荧光蛋白的表达情况并用G418筛选阳性细胞克隆AGS-P和AGS-pTP.免疫细胞化学、逆转录聚合酶链反应（RT-PCR）检测胃癌细胞株中TP蛋白及TP mRNA的表达情况,四甲基偶氮唑蓝（MTY）比色法检测上述三种胃癌细胞株对氟尿嘧啶类药物的敏感性.结果 构建了稳定表达TP和绿色荧光融合蛋白的胃癌细胞株AGS-pTP及稳定表达绿色荧光蛋白的胃癌细胞株AGS-p;免疫细胞化学显示AGS、AGS-P不表达TP蛋白,而AGS-pTP则强表达;RT-PCR检测显示AGS-pTP中TP mRNA表达水平（灰度比值,0.8090±0.0450）明显高于AGS（0.0490±0.0046）及AGS-P（0.0610±0.0069;P＜0.01）.AGS-pTP对去氧氟尿苷和卡倍他滨的敏感性明显增加,AGS-pTP对去氧氟尿苷和卡倍他滨的IC50值分别是AGS-P的1.7和2.2倍.相比AGS-P,AGS-pTP对5-FU的敏感性无明显改变.结论 提高胃癌细胞中TP表达水平能增强胃癌细胞对5-FU前体药卡倍他滨和去氧氟尿苷的敏感性,但并不影响胃癌细胞对5-FU的敏感性.

Objective To investigate the relationship between the expression of thymidine phosphorylase（TP）and the sensitivity of gastric carcinoma to 5-fluorouracil（5-FU）and its predrugs.Methods Gastric carcinom cell line AGS was transfected with recombinant plasmid pEGFP-N1-TP or control plasmid pEGFP-N1 by lipefectamin 2000.The expression of green fluorescence labeled protein was observed under fluorescence microscope.Positive clones AGS-P and AGS-pTP were selected by G418treatmenL Expression of TP protein and mRNA was detected by immunocytochemistry and RT-PCR.respectively.Drug sensitivitv to 5-FU and itg prodrugs was assessed by MTT assay.Results Cell clones with the expression of green fluorescent protein（AGS-P）and a clone with TP and green fluorescent fusion protein（AGS-pTP）were established.Immunostaining of TP protein was strongly positive in AGS-pTP and negative in AGS-P and AGS.The expression of TP mRNA was significantly higher in AGS-pTP（0.8090±0.0450）than that in AGS（0.0490±0.0046）and AGs-P（0.0610±0.0069;P〈0.01）.The sensitivity to doxifluridine and eapecitabine in AGS-pTP was significantly increased,as compared with that in AGS-p. IC50 values of AGS-pTP to doxifluridine and capecitabine were estimated 1.7 folds and 2.2 folds as much as that of AGS-P.respectively.The sensitivity to 5-FU was not different between AGS-pTP and AGS-P.Conclusions Enhancement of TP expression improves the sensitivitv of gastric carcinoma cells to doxifluridine and capecitabine.But it does not affect the sensitivity to 5-FU.