转染胸苷磷酸化酶基因对5’-脱氧氟尿苷抑制结肠癌细胞的影响  

Effect of thymidine phosphorylase cDNA transfection on the inhibition of human colon carcinoma cell Hne by 5＇-deoxy-S-fluorouridine

作　　者：高庆[1] 张继民[1] 刘剑[1] 王绮雯[2] 叶钿均[1] 刘影[3] 

机构地区：[1]广州医学院第二附属医院胃肠外科,510260 [2]广州医学院第二附属医院外科实验室,510260 [3]广州医学院第二附属医院核医学科,510260

出　　处：《中华胃肠外科杂志》2013年第4期370-375,共6页Chinese Journal of Gastrointestinal Surgery

基　　金：国家自然科学基金（81071883）

摘　　要：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TPcDNA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，RT—PCR法检测3组细胞TPmRNA表达；Westernblot法检测转染前后TP蛋白水平；MTF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11P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TPmRNA表达水平为空白对照组的（282．5＋86．8）倍（P〈O．01），而LOVO．载体组与对照组相比差异无统计学意义（P〉O．05）；Westernblot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）~mol／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）μmol／L，与对照组相比差异无统计学意义（P〉O．05）。培养基中分别加入0、500、1000和2000μmol／L的5＇-DFUR后，对照组培养基中分别检出0、2．10、3．13和7．19μmol／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40．02μmol／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TPcDNA能够明显提高LOVO细胞的TPmRNA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 MTI＂ 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.O1）, 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-transfeetants was（1087.7~89.1） p, mol/L, less than （1607.3~56.8） p, mol/L of parental cells significantly（P〈0.01）, while there was no significant difference between parental cells and vector-transfectants [ （1699.5~38.7） txmol/L,P〉O.05 ]. HPLC revealed that when medium was added with 0, 500, 1000, and 2000 Ixmol/L of 5＇-DFUR respectively, 0, 2.10, 3.13, and 7.19 Ixmol/L of 5-FU was found in the parental cells culture, while 0, 22.16, 30.94 and 40.02 Ixmol/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.

关 键 词：结直肠肿瘤 胸苷磷酸化酶 基因转染 5'-脱氧氟尿苷 氟尿嘧啶 

分 类 号：R735[医药卫生—肿瘤]