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

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）。在细胞裂解液中也检出有少量5-Fu，但其水平仅相当于培养基中的0．9％～4．2％。结论以慢病毒为载体将TP基因转染至人结肠癌细胞HT29和LS714T，能得到转染效率高和稳定传代的细胞株，并且2株细胞的TP蛋白和TPmRNA的表达水平明显增高。转染后在培养基中转化的5-Fu水平明显增高，使5．Fu和5’-DFUR对HT29和LS714T细胞的抗癌作用明显增强，但5’-DFUR增强效果更加明显。

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） μmol/L in its parent cells, respectively （ P 〈 0.05 ）. The IC50 values of 5-Fu of HT29-TP cells and its parents cells were （ 5.42±0.75 ） μmol/L and （ 14.19±0.97 ）μmol/L, respectively （ P 〈 0.05 ） , while （ 4.41 ±0.96 ） μmol/L in LS174T-TP cells and （ 16.42±2.12） μmot/L in its parents cells, respectively （ P 〈0. 05 ）. The HPLC results showed that the 5-Fu concentration detected from media contained a series of concentrations of 5＇-DFUR for culturing HT29-TP and LS174T-TP cells were 12.2 to 28.7-folds and 13.1 to 23.6-folds, respectively, higher than that in their parents cells, （P 〈 0.01 for all）. Otherwise, just a little of 5-Fu was detected in the two TP-transfected cells lysate, about 0.9% to 4.2% of 5-Fu detected in the media of the same cultured cells, whereas no 5-Fu was detected in the two parent cell lysates. Conclusions Transfection of TP eDNA into eolorectal cancer cell lines HT29 and LS174T with lentiviral vector can improve the expression of both TP mRNA and TP protein levels in passaged cells, enhance the conversion of 5-Fu from 5 ＇-DFUR in the medium, and result in an enhanced anticancer effect on these two cell lines.