中文摘要：

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.

英文摘要：

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 hig