基于基因转染法的胸苷磷酸化酶高表达结肠癌细胞株的建立

Enhancement of thymidine phosphorylase expression in the human colorectal cancer cell lines by transfected TP cDNA with lentiviral vector

目的构建表达高度胸苷磷酸化酶(TP)活性并可以稳定传代的结肠癌细胞株。方法合成包含TP c DNA全长的真核细胞表达载体,用慢病毒包装后转染人结肠癌细胞株SW480、LOVO、HT29和LS174T,流式细胞仪检测转染效率,实时荧光定量RT-PCR方法检测TP的m RNA表达,蛋白印迹法(Western blot)检测TP蛋白表达。结果转染TP c DNA后,SW480、LOVO、HT29与LS174T在传代5代后转染效率仍稳定在95%左右。与未转染TP基因的亲代细胞相比,SW480-TP、LOVO-TP、HT29-TP与LS174T-TP的TP m RNA的表达分别提高了(694.56±171.53)、(282.46±86.85)、(8.45±0.15)和(2 615.02±253.97)倍,差异有统计学意义(P<0.05);并且TP蛋白的表达水平也显著提高。结论慢病毒载体能够高效地将TP c DNA转染至人结肠癌细胞SW480、LOVO、HT29和LS174T中,所得克隆能够稳定传代,并能显著提高4株结肠癌细胞TP的m RNA和蛋白表达水平。

Objective To build the human colorectal cancer cell lines with the high expression of thymidine phosphorylase（ TP） by transfected TP c DNA with lentiviral vector. Methods Human colorectal cancer cell lines SW480,LOVO,HT29 and LS174 T were transfected with human lentiviral vector TP c DNA by p Lenti6. 3_MCS_IRES2- EGFP.The transfection efficiency was analyzed by flow cytometer,the m RNA expression of TP was detected by RT- PCR and the TP level was detected by Western blot. Results The stabilized transfection efficiency was about 95% past 5 generations.Comparing with parental SW480,LOVO,HT29 and LS174 T,the m RNA expression of SW480- TP,LOVO- TP,HT29-TP and LS174T- TP were significantly up- regulated by（ 694. 56 ± 171. 53）,（ 282. 46 ± 86. 85）,（ 8. 45 ± 0. 15） and（ 2 615. 02 ± 253. 97） folds,respectively（ P〈0. 05）. The Western blot showed that the TP levels of SW480- TP,LOVO- TP,HT29- TP and LS174T- TP were obvious up- regulated. Conclusion Stabilized transfections of 4 cell lines with a high TP expression is successfully constructed by lentiviral vector,with significantly up- regulation of TP in m RNA and protein levels.