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目的 探讨转染胸苷磷酸化酶(TP)cDNA对5’-脱氧氟尿苷(5’-DFUR)抑制人结肠癌细胞LOVO作用的影响.方法 将TP cDNA序列克隆以慢病毒表达载体包装后转染人结肠癌细胞LOVO(LOVO-TP组),另设空白对照组和LOVO-载体组.以流式细胞仪检测3组细胞转染效率,RTPCR法检测3组细胞TP mRNA表达;Western blot法检测转染前后TP蛋白水平；MTT法检测转染前后LOVO对5’-DFUR药物敏感性的变化；高效液相色谱法(HPLC)检测3组细胞转化不同浓度5'-DFUR生成5-FU量的情况.结果 转染TP基因并传代5代后,LOVO细胞的转染效率在95％左右.转染TP基因后,LOVO-TP组的TP mRNA表达水平为空白对照组的(282.5±86.8)倍(P＜0.01),而LOVO-载体组与对照组相比差异无统计学意义(P＞0.05)；Western blot检测显示,LOVO-TP组的TP蛋白表达明显高于对照组和LOVO-载体组.5’-DFUR对LOVO细胞半数有效剂量(IC50)对照组为(1607.3±56.8) μmol/L,明显高于LOVO-TP组的(1087.7±89.1) μmol/L(P＜0.01)；而LOVO-染载体组为(1699.5±38.7)tμmol/L,与对照组相比差异无统计学意义(P＞0.05).培养基中分别加入0、500、1000和2000 μmol/L的5'-DFUR后,对照组培养基中分别检出0、2.10、3.13和7.19 μmol/L的氟尿嘧啶(5-FU)；而在LOVO-TP组的细胞培养基中则分别检出0、22.16、30.94和40.02 μmol/L的5-FU;而LOVO-载体组的培养基中则几乎无5-FU检出.结论 转染TP cDNA能够明显提高LOVO细胞的TP mRNA及TP蛋白表达水平,使细胞外5’-DFUR转化为5-FU增多,明显提高5’-DFUR对LOVO的细胞毒性作用.

Objective To investigate the inhibiting impact of 5'-deoxy-5-fluorouridine(5'-DFUR)on human colon carcinoma cell line LOVO after transfection of thymidine phosphorylase (TP) cDNA.Methods TP cDNA was transfected into human colon carcinoma cell line LOVO with lentiviral vector pLenti6.3_MCS_IRES2-EGFP,and the transfection efficiency was analyzed by flow cytometry.TP mRNA and protein expressions were detected by RT-PCR and Western blotting respectively.The IC50 of 5'-DFUR on TP-transfected LOVO and parental cell were evaluated by MTT assay.The volumes of 5-FU converted from 5'-DFUR in media,where TP-transfected and parental LOVO were cultured,were detected by HPLC.Results The stable transfectants passed 5 generations were obtained and the transfection rate was 95％.Compared with parental cell,the RQ values of mRNA expression in TP-transfected LOVO was(282.5±86.8) folds higher significantly(P＜0.01),also the TP protein expression of TP-transfected LOVO was obviously up-regulated as compared to parental cells.The IC50 value of 5'-DFUR of TP-transfectants was(1087.7±89.1) μmol/L,less than (1607.3±56.8) μmol/L of parental cells significantly(P＜0.01),while there was no significant difference between parental cells and vector-transfectants [(1699.5±38.7) μmol/L,P＞0.05].HPLC revealed that when medium was added with 0,500,1000,and 2000 μmol/L of 5'-DFUR respectively,0,2.10,3.13,and 7.19 μmol/L of 5-FU was found in the parental cells culture,while 0,22.16,30.94 and 40.02 μmol/L of 5-FU was found in TP-transfectants culture,but no 5-FU was found in the vector-transfectants culture.Conclusion TP cDNA transfection into LOVO can up-regulate the TP mRNA and protein expressions,increase the 5-FU converted from 5'-DFUR,and enhance the cytotoxic effect of 5'-DFUR on the LOVO cells.