中文摘要：

目的 利用慢病毒载体将胸苷磷酸化酶（TP）基因转染人结肠癌细胞株LOVO,然后应用干扰素-α2a（INF-α2a）处理转染前后细胞,分别测定5＇-脱氧氟尿苷（5＇-DFUR）和5-氟尿嘧啶（5-FU）对LOVO抗肿瘤效应的影响.方法 构建包含TP基因的慢病毒载体,转染结肠癌细胞株LOVO,以流式细胞技术和免疫荧光法检测在第5代LOVO中的转染效率;加INF-α2a培养24h后分别应用RT-PCR和Wester blot检测细胞的TP mRNA和TP蛋白表达;MTS法检测5＇-DFUR和5-FU对各组细胞的半数抑制浓度（IC50）;高效液相色谱法检测各组细胞转化5＇-DFUR为5-FU的量.结果 转染TP基因的效率达95％.LOVO＋INF-α2a组的TP mRNA表达是LOVO组的1.87倍,而LOVO-TP组和LOVO-TP＋ INF-α2a组TPmRNA表达则比LOVO组分别增加了18.56倍和59.61倍（P＜0.01）.LOVO-TP＋ INF-α2a组和LOVO-TP组蛋白表达水平比LOVO组分别增加242.23倍和197.69倍（P＜0.01）.5＇-DFUR对LOVO-TP组细胞的IC50值仅为LOVO组细胞的37.2％,而LOVO-TP＋INF-α2a组的IC50则是LOVO组的18.2％.LOVO-TP组在加入100、200、400 μmol/L5＇-DFUR的培养基中检测的5-FU浓度比LOVO组分别增高了26.13％、33.23％、32.95％,而LOVO-TP＋ INF-α2a组比LOVO组分别增高了49.94％、61.66％、72.14％. 结论 转染TP eDNA能够明显提高LOVO细胞的TP mRNA及TP蛋白的表达水平,而INF-α2a则进一步增强这种上调作用.增高的TP表达使细胞外5＇-DFUR转化为5-FU含量增加,明显提高5＇-DFUR的细胞毒性.

英文摘要：

Objective Thymidine phosphorylase （TP） cDNA was transfected into colorectal cancer cell lines LOVO with the lentiviral vector,the anticancer effeciency of 5＇-deoxy-5-fluorouridine （5＇-DFUR）and 5-fluorouracil （5-FU） on LOVO cells were evaluated.Methods TP cDNA were transfected into LOVO cell line with the lentiviral vector pLenti6.3_MCS_IRES2-EGFP,and the transfection efficiency was analyzed by flow cytometer and immunohistochemistry.Cells were divided into six groups：LOVO,LOVO-TP,LOVO-control,LOVO ＋ INF-α2a,LOVO-TP ＋ INF-α2a,LOVO-control ＋ INF-α2a.TP protein expression and the relative quantitative analysis for TP mRNA in transfections cells were detected by Western blot and RT-PCR respectively.Volumes of converted 5-FU from either in the medium containing different concentration of 5＇-DFUR,in which all cells were cultured,or in cells lysate,were detected by high performance liquid chromatography （HPLC）.Results The transfection efficiency of TP cDNA in LOVO cells was 95%.The Mean gray value of TP protein expression in LOVO-TP and LOVO-TP ＋ INF-α2a were 198.15 folds and 243.22 folds higher than LOVO cell,respectively （P 〈 0.01）.The RQ values of TP mRNA expression in LOVO-TP and LOVO-TP ＋ INF-α2a were also 18.56 folds and 59.61 folds higher than wild LOVO cell,respectively （P 〈 0.01）.The IC50 values of 5＇-DFUR on LOVO-TP and LOVO-TP ＋ INF-α2a were 1 660 μ mol/L and 813 μ mol/L,respectively,significantly lower than 4 462.59 μ mol/L in wild LOVO （P 〈 O.01）.The 5-FU volumes detected from media contained series concentration of 5＇-DFUR for culturing LOVO-TP and LOVO-TP ＋ INF-α2a were 2.0-5.3 folds,and 2.9-10.4 folds more than wild LOVO,respectively （P 〈 0.01）.Conclusions Transfected TP cDNA into colorectal cancer cell line LOVO with lentiviral vector increases the expression of TP mRNA and TP protein and the amount of 5-FU converted from 5＇-DFUR,enhancing its anticancer effect.