胞嘧啶脱氨酶和胸苷激酶融合双自杀基因系统对膀胱癌细胞体外杀伤作用

Effect of Cytocine Deaminase-Thymidine Kinase Double Suicide Fusion Gene Therapy on the Growth of Murine Bladder Carcinoma Cells

目的探讨胞嘧啶脱氨酶(CD)和胸苷激酶(TK)融合双自杀基因系统对膀胱癌细胞的杀伤作用。方法利用含有CD-TK双自杀融合基因及绿色荧光蛋白(GFP)基因的复制缺陷腺病毒感染膀胱癌细胞株Mb49细胞,RT-PCR检测CD及TK表达,MTT法测定感染病毒Mb49细胞对GCV和/或5-FC敏感性及旁观者效应。结果RT-PCR可检测到腺病毒感染的Mb49细胞中CD及TK表达,腺病毒感染的Mb49细胞对GCV和/或5-FC敏感性增强,且细胞存活率随前药浓度增加而明显降低,联合应用杀伤效果更强。在混合培养细胞中转染细胞为10%时,GCV组、5-FC组、GCV+5-FC组细胞存活率分别为(79.55±0.88)%、(77.17±3.38)%、(49.21±1.78)%,有较强的旁观者效应。结论腺病毒载体能有效转导CD-TK融合双自杀基因在膀胱癌细胞中表达,CD-TK融合双基因系统对膀胱癌细胞有更强杀伤效果和更明显的旁观者效应,优于单基因系统,值得进一步研究。

Objective To investigate the effect of cytocine deaminase-thymidine kinase double suicide fusion gene therapy on the in vitro growth of murine bladder carcinoma cells. Methods Murine bladder carcinoma Mb49 cells were infected by replication-defective recombinant adenovirus containing cytoeine deaminase - thymidine kinase double suicide fusion gene and green fluorescent protein （GFP） gene. The expressions of CD and TK gene were examined by reverse transeriptase-polymerase chain reaction （RT-PCR）. The cytotoxic effect and ＂bystander effect＂ of GCV or/and 5-FC on infected cells were determined by MTT assay. Results The double suicide fusion gene was successfully transferred and expressed by Mb49 cells. In the presence of prodrugs, the survival rate of the infected Mb49 cells was markedly decreased and the ＂bystander effect＂ was apparent. The double suicide gene system was proven to be more effective than the single suicide gene system.Conclusion CD-TK double suicide fusion gene can be transferred efficiently into bladder carcinoma cells and reduces the survival of tumour cells.