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目的:构建前列腺特异性膜抗原(PSMA)启动子调控表达的FCY1/TK双自杀基因表达载体,并观察单、双自杀基因对前列腺癌细胞LNCaP的杀伤作用.方法:通过PCR方法扩增出基因组DNA中PSMA启动子,利用基因重组的方法,替换真核表达载体pIRES中的CMV启动子.测序正确后,再将自杀基因FCY1和HSVTK分别克隆于载体pIRES的两个多克隆位点,构建真核表达载体pPSMAIRESFCY1TK.将该载体转染LNCaP细胞,用RTPCR的方法检测其转录,用MTT法检测前体药物5氟胞嘧啶、丙氧鸟苷单一或联合使用对转染后的LNCaP细胞的杀伤作用.结果:PCR扩增出长1400bp的PSMA启动子片段,经克隆至pIRES后酶切鉴定证实,并测序表明序列与GenBank(AccessionNumberAF007544)报道的一致.pPSMAIRESFCY1TK经脂质体转染LNCaP细胞后,RTPCR检测到FCY1和TK基因在该细胞中均有转录.MTT法检测表明5FC与GCV联合使用,其细胞生长抑制率明显高于单独使用5FC,GCV(P<0.05).结论:pPSMAIRESFCY1TK的双自杀基因体系对前列腺癌细胞LNCaP细胞的杀伤作用,明显优于单自杀基因体系.
Objective: To construct FCY1 / TK dual suicide gene expression vector regulated by prostate specific membrane antigen (PSMA) promoter and to observe the killing effect of single and double suicide genes on LNCaP cells.Methods: Genomic DNA was amplified by PCR DNA in the PSMA promoter, using gene recombination method to replace CMV promoter in the eukaryotic expression vector pIRES correct sequencing, and then suicide genes FCY1 and HSVTK were cloned in the vector pIRES two multiple cloning sites, the construction of true The vector pPSMAIRESFCY1TK was transfected into LNCaP cells, and the transcription of LNCaP cells was detected by RTPCR. The cytotoxicity against LNCaP cells transfected with 5-fluorocytosine and ganosine alone or in combination was detected by MTT assay .Results: The 1400 bp long promoter fragment of PSMA was amplified by PCR and confirmed by restriction enzyme digestion after cloning to pIRES, and sequencing showed that the sequence was consistent with that reported by GenBank (Accession Number NA007544). After transfection of LPSaIRESFCY1TK into LNCaP cells by liposome, RTPCR detected that both FCY1 and TK genes were transcribed in this cell.MTT assay showed that the combination of 5FC and GCV had a significantly higher rate of cell growth inhibition than that of 5FC and GCV alone ) Conclusion: The killing effect of double suicide gene system of pPSMAIRESFCY1TK on prostate cancer cell LNCaP cells is obviously better than single suicide gene system.