Gao Qing, Zhang Ji-min, Liu Jian, Wang Qi-wen, Ye Dian-jun, Liu Ying
Department of Gastrointestinal Surgery, Guangzhou Medical College, Guangzhou, China.
Zhonghua Wei Chang Wai Ke Za Zhi. 2013 Apr;16(4):370-5.
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.
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.
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.
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.
探讨转染胸苷磷酸化酶（TP）cDNA后5'-脱氧-5-氟尿苷（5'-DFUR）对人结肠癌LOVO细胞系的抑制作用。
用慢病毒载体pLenti6.3_MCS_IRES2-EGFP将TP cDNA转染到人结肠癌LOVO细胞系，通过流式细胞术分析转染效率。分别用RT-PCR和Western印迹法检测TP mRNA和蛋白表达。用MTT法评估5'-DFUR对转染TP的LOVO细胞和亲本细胞的半数抑制浓度（IC50）。用高效液相色谱法（HPLC）检测培养转染TP的LOVO细胞和亲本LOVO细胞的培养基中由5'-DFUR转化而来的5-氟尿嘧啶（5-FU）的量。
获得了传代5次的稳定转染细胞株，转染率为95%。与亲本细胞相比，转染TP的LOVO细胞中mRNA表达的相对定量（RQ）值显著升高（282.5±86.8）倍（P<0.01），转染TP的LOVO细胞中TP蛋白表达也明显高于亲本细胞。转染TP的细胞对5'-DFUR的IC50值为（1087.7±89.1）μmol/L，明显低于亲本细胞的（1607.3±56.8）μmol/L（P<0.01），而亲本细胞与转染载体的细胞之间无显著差异[（1699.5±38.7）μmol/L，P>0.05]。HPLC显示，当培养基中分别加入0、500、1000和2000μmol/L的5'-DFUR时，亲本细胞培养物中检测到0、2.10、3.13和7.19μmol/L的5-FU，转染TP的细胞培养物中检测到0、22.16、30.94和40.02μmol/L的5-FU，而转染载体的细胞培养物中未检测到5-FU。
将TP cDNA转染到LOVO细胞中可上调TP mRNA和蛋白表达，增加由5'-DFUR转化而来的5-FU的量，并增强5'-DFUR对LOVO细胞的细胞毒作用。
