转染胸苷磷酸化酶基因对5＇-脱氧氟脲苷抗结肠癌细胞株活性的影响

Enhanced anticancer effects of 5′-DFUR on colorectal cancer cell lines SW480 and LOVO by transfection with thymidine phosphorylase cDNA

目的 探讨人结肠癌细胞系SW480和LOVO细胞株转染胸苷磷酸化酶（TP）基因后转化5′-脱氧氟脲苷（5′-DFUR）为氟尿嘧啶（5-FU）能力的变化及5′-DFUR抗癌细胞株活性的变化.方法 构建包含TP cDNA的真核表达载体,用慢病毒包装后转染结肠癌细胞株SW480和LOVO.转染5代后以免疫荧光法和流式细胞技术检测转染效率;RT-PCR和Western blot法分别检测2株细胞的TP mRNA和TP蛋白表达.然后以高效液相色谱法（HPLC）检测2株细胞转染前后转化5′-DFUR为5-FU的量;MTT法检测5′-DFUR对2株细胞转染前后半数抑制浓度（IC50）.结果 转染TP基因后SW480-TP与LOVO-TP 5代细胞转染效率稳定在95％左右.2株转染细胞的TP mRNA表达分别比野生型细胞增加（695±172）倍（t=-7.00,P=0.002）和（282±87）倍（t=-5.61,P=0.030）,Western blot显示TP蛋白表达也明显增强.转染后的SW480-TP与LOVO-TP在培养基中分别使5′-DFUR转化为5-FU的量增加（t=19.406 ～66.921,P＜0.01）.5′-DFUR对SW480的IC50由（1641±53） μmol/L降至（587±17） μmol/L（t=-32.59,P＜0.01）;对LOVO的IC50由（1607±57） μmol/L降至（1088±89） μmol/L（t=-8.52,P＜0.01）.结论 慢病毒载体能够高效地将TP cDNA转染至人结肠癌细胞SW480和LOVO并稳定传代,转染后2株细胞的TP mRNA和TP蛋白表达明显增高,使5′-DFUR转化为5-FU的量增加,对结肠癌细胞株SW480和LOVO的抑制作用增强.

Objectives To study the change of ability to transform from 5＇-deoxy-fluorouracil monophosphate （5＇-DFUR） to fluorouracil （5-FU） in human colon cancer cell lines SW480 and LOVO which transfected with thymidine phosphorylase （TP） gene. And to discuss the anti-cancer activity of 5＇-DFUR to SW480 and LOVO cells. Methods TP cDNA were transfected into human colorectal cancer cell lines SW480 and LOVO with the lentiviral vector, pLenti6. 3_MCS_IRES2-EGFP. The transfection efficiency was analyzed by flow cytometer, the mRNA expression of TP was detected by RT-PCR, and the TP protein expression was detected by Western blot, and the volumes of 5-FU converted from 5＇-DFUR both in 2 ceils and medium were detected by high performance liquid chromatography （HPLC）. The 50% inhibitory concentration （ IC50 ） of 5＇-DFUR on these 2 colon cancer cell lines both wild type and TP-transfected cells were evaluated by MTI＂ assay. Results The colorectal cancer cell lines SW480 and LOVO transfected with human TP cDNA were monitored 5 generations, and the transfections efficiency rate wea about 95%. Compared with wild type cell SW480 and LOVO, the RQ values of mRNA expression of SW480-TP and LOVO-TP were （695＋ 171） folds （t = -7.00, P =0.002） and （282 ＋87） folds （t = -5.61, P = 0. 030）, respectively. Also TP protein expression in SW480-TP and LOVO-TP were higher than their parent cells shown by Western blot. The volume of 5-FU converted from 5＇-DFUR in the medium cultured SW480- TP and LOVO-TP were increased compared with their parent ceils, respectively （ t = 19. 406-66. 921, P 〈 0. 01 ）, whereas few of 5-FU was detected both in wild, and TP-transfected cells. After transfected with TP cDNA, the IC50 of 5＇-DFUR on SW480-TP and LOVO-TP were （ 587 ＋ 17 ） μmol/L and （ 1088 ＋ 89） μmol/L respectively, and there were significantly less than their parent cells （ t = - 32. 59 and - 8.52, P 〈 0.01 ）. Conclusions The stabilized transfections of SW480 and LOVO with higher TP expression could be built with lentiviral vector. Transfected TP cDNA into SW480 and LOVO, could improve the expression both of TP mRNA and TP protein, increase the volume of 5-FU converted from 5＇-DFUR in medium, and result in an enhancement of anticancer effect on these 2 cells.