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在离体条件下探讨CD/5 FC自杀基因治疗系统对恶性人脑胶质瘤细胞的抗癌作用。通过将CD基因插入真核表达载体pcDNA3.0多克隆位点构建pCMVCD表达质粒 ,采用限制性内切酶消化鉴定所构建的质粒 ,并进行CD基因测序。采用LipofectAMINE2 0 0 0脂质体介导法将CD基因转染U2 5 1恶性人脑胶质瘤细胞 ,G4 18筛选获得抗性克隆 (取名为U2 5 1/CD细胞 ) ,使用不同浓度的 5 FC作用于U2 5 1/CD细胞 ,MTT法测定活性细胞比率。采用高效液相色谱法 (HPLC)检测 5 FC培养液内 5 FU的浓度。结果如下 :真核表达质粒pCMVCD构建成功 ,并通过酶切鉴定。U2 5 1细胞获得了质粒的成功转染。未转染的U2 5 1细胞对 5 FC不敏感 ,IC50 约为 6 5 0 0 μmol/L ,而转染基因后IC50 约为 10 μmol/L。基因转染使G4 18抗性细胞(U2 5 1/CD细胞 )对 5 FC高度敏感。并且加入不同浓度的 5 FC后 ,U2 5 1/CD细胞培养液内均能检测到 5 FU。实验结果表明 :CD/5 FC系统可以用于胶质瘤的治疗 ,CD基因修饰U2 5 1细胞及其表达的离体研究为胶质瘤基因治疗的在体研究提供依据。
To investigate the anticancer effect of CD / 5 FC suicide gene therapy system on malignant human glioma cells in vitro. The pCMVCD expression plasmid was constructed by inserting the CD gene into the multi-cloning site of the eukaryotic expression vector pcDNA3.0. The constructed plasmid was identified by restriction endonuclease digestion and the CD gene was sequenced. The CD gene was transfected into U2 5 1 human malignant glioma cells by LipofectAMINE 2000 microdissection. The resistant clones (named as U2 5 1 / CD cells) were screened by G4 18, 5 FC on U2 5 1 / CD cells, the ratio of active cells was determined by MTT method. The concentration of 5 FU in 5 FC medium was detected by high performance liquid chromatography (HPLC). The results are as follows: The eukaryotic expression plasmid pCMVCD was successfully constructed and identified by restriction enzyme digestion. U2 5 1 cells obtained a successful transfection of plasmids. Untransfected U2 5 1 cells were insensitive to 5 FC with an IC50 of about 6500 μmol / L, whereas IC50 was about 10 μmol / L after transfection. Gene transfection allowed G4 18 resistant cells (U2 5 1 / CD cells) to be highly sensitive to 5 FC. After adding 5 FC with different concentrations, 5 FU could be detected in U2 5 1 / CD cell culture medium. The experimental results show that the CD / 5 FC system can be used for the treatment of gliomas. The in vitro study of CD gene-modified U251 cells and their expression provides the basis for in vivo study of gene therapy of glioma.