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目的观察联合应用自杀基因/前药系统和放疗对人骨肉瘤细胞的杀伤作用。方法以大肠杆菌JM107为模板,PCR扩增出CD基因,测序并将其插入真核表达质粒pEGFP-C1中,构建EGFPCD融合基因。用DOTAP将分别含有CD和HSV-TK的两种质粒pEGFP-CD和tgCMVHyTK同时导入人骨肉瘤细胞SAOS-2,经G418和潮霉素B共同筛选后,于荧光显微镜下观察阳性克隆细胞株SAOS-2CDTK。运用PCR和Northernblot方法检测SAOS-2CDTK细胞中的CD和HSV-TK基因及其表达。用MTT法检测单独或联合应用两种前药(GCV、5-FC)对SAOS-2CDTK细胞的存活率、“旁观者效应”以及细胞对放疗敏感性的影响。结果测序图和酶切鉴定证明CD基因的克隆和含EGFPCD融合基因的重组真核表达质粒pEGFP-CD的构建是成功的。荧光显微镜观察SAOS-2CDTK细胞胞质呈现绿色荧光。PCR和Northernblot分析均表明SAOS-2CDTK细胞中含有CD和HSV-TK两个基因,并获得mRNA水平的表达。与单一前药相比,联合应用两种前药时,SAOS-2CDTK细胞的存活率下降,“旁观者效应”及细胞对放疗的敏感性增强。结论联合应用两种自杀基因/前药系统和放疗可增强细胞毒作用,为骨肉瘤的治疗提供了一条新的有效途径。
Objective To observe the killing effect of combined suicide gene / prodrug system and radiotherapy on human osteosarcoma cells. Methods The E. coli JM107 was used as a template to amplify the CD gene by PCR. The gene was inserted into eukaryotic expression plasmid pEGFP-C1 to construct EGFPCD fusion gene. The two plasmids pEGFP-CD and tgCMVHyTK containing CD and HSV-TK, respectively, were simultaneously introduced into human osteosarcoma cell line SAOS-2 by DOTAP. After screened by G418 and hygromycin B, the positive clones were observed under a fluorescence microscope. 2CDTK. The CD and HSV-TK genes and their expression in SAOS-2 CDTK cells were detected by PCR and Northern blotting. The survival rate of SAOS-2CDTK cells, “bystander effect” and the effect of cells on radiosensitivity of two kinds of prodrugs (GCV, 5-FC) alone or in combination were detected by MTT assay. Results Sequencing and restriction analysis proved that the cloning of CD gene and the construction of recombinant eukaryotic expression plasmid pEGFP-CD containing EGFPCD fusion gene were successful. Fluorescence microscopy showed that SAOS-2CDTK cells showed green fluorescence in the cytoplasm. Both PCR and Northern blot analysis showed that SAOS-2CDTK cells contained both CD and HSV-TK genes and their mRNA expression levels were obtained. Compared with a single prodrug, the combination of two prodrugs, SAOS-2CDTK cell survival decreased, “bystander effect” and cell sensitivity to radiotherapy increased. Conclusion Combined application of two suicide gene / prodrug systems and radiotherapy can enhance cytotoxicity and provide a new and effective approach for the treatment of osteosarcoma.