慢病毒转染胸苷磷酸化酶基因增强5’-脱氧氟尿苷抗结肠癌细胞活性  

Transfection of thymidine phosphorylase cDNA with lentiviral vector enhances the anticancer effect of S＇-deoxy-5-fluorouridine on colorectal cancer cell lines HT29 and LS174T

作　　者：叶钿均[1] 王绮雯[2] 张继民[1] 刘奇[1] 

机构地区：[1]广州医科大学附属第二医院胃肠外科,510260 [2]广州医科大学附属第二医院外科实验室,510260

出　　处：《中华肿瘤杂志》2015年第1期18-24,共7页Chinese Journal of Oncology

基　　金：国家自然科学基金（81071883）

摘　　要：目的探讨5’-脱氧氟尿苷（5’-DFUR）和氟尿嘧啶（5-Fu）对人结肠癌细胞株HT29和LS174T转染胸苷磷酸化酶（TP）基因前后抗癌效应的变化。方法构建包含TP基因的慢病毒载体，转染结肠癌细胞株HT29和HLS174T。传代5代后，以流式细胞术检测转染效率。免疫组织化学染色和Westernblot检测转染TP基因前后HT29和LS174T细胞中TP蛋白的表达，逆转录聚合酶链反应（RT—PCR）法检测细胞中TPmRNA的表达水平，四唑氮化合物法检测5’-DFUR和5-Fu对转染TP基因前后细胞株的半数抑制浓度（IC50），高效液相色谱（HPLC）法检测HT29和LS174T在细胞转染rrP基因前后转化5’-DFUR为5-Fu的水平。结果转染TP基因的HT29和LS174T细胞传代5代后，转染效率稳定在约95．0％。转染TP基因的HT29和LS174T细胞中TP表达阳性，而野生型细胞和仅转染病毒载体细胞中TP表达阴性；Westernblot结果显示，HT29和HLS174T细胞中’rP蛋白的表达明显增强。RT-PCR结果显示，转染TP基因的HT29和LS174T细胞中TPmRNA的相对表达水平分别为8．45±0．15和2615．02±253．97，与野生型细胞比较，差异均有统计学意义（均P〈0．01）。5’-DFUR对转染TP基因的HT29-TP细胞和野生型细胞的IC50分别为（14．33±0．74）μmol／L和（707．66±5．66）μmol／L（P〈0．05）；对转染TP基因的LS174T—TP细胞和野生型细胞的IC50分别为（0．59±0．11）μmol／L和（239．20±21．83）μmol／L（P〈0．05）。5-Fu对转染TP基因的HT29-TP细胞和野生型细胞的Ic50分别为（5．42±0．75）μmol／L和（14．19±0．97）μmol／L（P〈0．05）；对转染TP基因的LS174T—TP细胞和野生型细胞的Ic。分别为（4．41±0．96）μmol／L和（16．42±2．12）μmol／L（P〈0．05）。转染TP基因后的HT29和HLS174T细胞培养基中，则检出大量转化的5-Fu，较转染前分别增加了12．2～23．6倍，差异均有统计学意义（均P〈0．01）。�Objective To explore the changes of anticancer efficiency of 5＇-deoxy-5-fluorouridine （5＇-DFUR） and 5-fluorouracil （5-Fu） in colorectal cancer cell line HT29 and LS174T cells after transfection of thymidine phosphorylase （TP） cDNA with a lentiviral vector. Methods TP cDNA was transfected into human colorectal cancer cell lines HT29 and IS174T with the lentiviral vector pLenti6.3- MCS-IRES2-EGFP, and the transfection efficiency of the two cell lines passed 5 generations was analyzed by flow cytometry. The expression of TP protein and the relative quantitative expression of TP mRNA in these 2 cell lines were detected by Western blot and RT-PCR, respectively. The 50% inhibitory concentration （ IC50 ） of 5＇-DFUR and 5-Fu in both HT29 and LSI74T parent cells and TP-transfected cells were assessed by MTS assay. Finally, the concentration of converted 5-Fu was detected by high performance liquid chromatography （HPLC） either in the medium containing a series of concentrations of 5＇-DFUR, in which HT29/HT29-TP or LS174T/LS174T-TP cells were cultured, or in the cell culture lysates. Results The HT29 and ES174T cells transfected with human TP cDNA were monitored for 5 generations, and their transfection efficiency was about 95.0%. Immunohistochemical staining showed that both the parent cells and TP-transfected HT29 and LS174T cells were TP-positive, while vector-transfected cells were TP- negative. Western blotting showed that the TP protein expression in HT29-TP and LS174T-TP cells were significantly increased compared with that in their parents cells. The relative quantity （RQ） values of TP mRNA in HT29-TP and LS174T-TP cells were 8.45±0. 15 and 2 615.02±253.97, respectively, which were significantly higher than that in their parents cells （ P 〈0.01）. The IC50 values of 5＇-DFUR on HT29- TP cells and its parents cell were （ 14.33±0.74 ） i^mol/L and （707.66±5.66）μmol/L, respectively （P〈0.05）, while （0.59±0. 11） txmolfL in LS174T-TP ceils and （239.20±21.83�

关 键 词：结直肠肿瘤 胸苷磷酸化酶 转染 5'-脱氧氟尿苷 氟尿嘧啶 

分 类 号：R735.35[医药卫生—肿瘤]