摘要
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 investigate the killing effect of combined use of double suicide gene /prodrug system and radiotherapy on o steosarcoma cells.Methods CD gene was amplified from E.Coli JM107by PCR.The PCR product was inse rted into sequencing plasmid pUC19a nd sequenced,then CD gene and eukaryotic expression plasmid p EGFP -C1were ligated to construct th e EGFPCD fusion gene expressed in the same transcription unit with gen e recombinant technology.The positive recombinants were identified by digestion of restriction endonucle ase.The plasmids pEGFP -CD and tgCMVHyTK,carrying CD and HSV -TK gene respectively,were intruduced into osteosarcoma cell SAOS -2by DOTAP.The selected pressure was maintained with G418and Hygromycin B.The positive clone concomitantly expressed two genes was named by SAOS -2CDTK cell.Co-expression of t he EGFPCD and HyTK gene in SAOS -2CDTK cells was detected by PCR and Northern blot.MTT assay was u sed to detect the cytotoxicity,bystander effect and radiosensitivity o f SAOS -2CDTK cells exposed to single a nd double prodrugs.Results Sequencing and restriction endonu-clease digestion proved the success of CD gene clone and construction of r ecombinant eukaryotic expression plasmid pEGFP -CD harboring EGFPCD f usion gene.The green fluorescence c ould be observed in cytoplasm of SAOS -2CDTK cells under fluoresce nce phase-contrast microscopy.Two prominent bands of 594bp CD and395bp HSV -TK fragments were generated in one PCR reaction using DNA from S AOS -2CDTK cells as template.Hybridization of CD and HS V -TK probes with total RNA from SAOS -2CDTK cells showed bands of the expected size,2kb(1.28kb CD +0.7kb EGFP)and 2.09kb(1.03kb HSV -TK +1.06kb Hy )respectively.A combination of GCV a nd 5-FC resulted in 10.2%survival ra te of SAOS -2CDTK that was six-fold less than 9μg GCV(67.4%),seven-fold less than 100μg 5-FC(77.1%).When the mixed(20%SAOS -2CDTK and 80%SAOS -2)cells were exposed to 5-FC,GCV and both prodrugs,the survival rate of SAOS -2cells were 57.6%,10.4%and0respectively.Surviving fraction of SAOS -2CDTK cells treated with GCV and HSV -TK for 24hours prior to irradiation dramatically decreased than single prodrug.Con-clusion Combining suicide gene therapy with radiotherapy could result in enhanced cytotoxicity,thereby provide a new and effective means for treatment of osteosarcoma.[
出处
《中华骨科杂志》
CAS
CSCD
北大核心
2002年第7期427-431,共5页
Chinese Journal of Orthopaedics
基金
国家自然科学基金资助项目(39970289)
关键词
骨肉瘤
基因疗法
放射疗法
胞嘧啶
脱氨酶类
胸苷激酶
Osteosarcoma
Gene therapy
Radiotherapy
Cyto-sine
Ammonia-lyases
Simplexviru s
Thymidine kinase
