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

摘要

Objective To build the human colorectal cancer cell lines with the high expression of thymidine phos -phorylase (TP) by transfected TP cDNA with lentiviral vector .Methods Human colorectal cancer cell lines SW480, LOVO, HT29 and LS174T were transfected with human lentiviral vector TP cDNA by pLenti 6.3_MCS_IRES2 -EGFP. The transfection efficiency was analyzed by flow cytometer , the mRNA 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 LS174T, the mRNA 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 SW 480 -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 mRNA and protein levels.%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 cDNA 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 mRNA 表达，蛋白印迹法（Western blot）检测 TP 蛋白表达。结果转染 TP cDNA 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 mRNA 的表达分别提高了（694．56±171．53）、（282．46±86．85）、（8．45±0．15）和（2615．02±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 cDNA 转染至人结肠癌细胞 SW480、LO－VO、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 mRNA 和蛋白表达水平。