酵母菌胞嘧啶脱氨酶/5-氟胞嘧啶基因治疗系统对K562B细胞杀伤效应的初步研究

Study on killing efficacy of yeast cytosine deaminase /5 - fluorocytosine gene therapy system on K562B cell line

目的 :克隆酵母菌胞嘧啶脱氨酶 (YCD)基因 ,观察 YCD/ 5氟胞嘧啶 (YCD/ 5 - FC)系统对转基因肿瘤细胞的杀伤效应。 方法 :扩增 YCD基因并构建 YCD基因重组逆转录病毒载体 ;载体转染包装细胞获得高滴度病毒并转染高致瘤细胞K5 6 2 B,筛选并鉴定阳性转基因克隆 ;以 MTT法检测 5 - FC对 YCD转基因细胞的杀伤效应 ,测定转基因细胞裂解产物对5 - FC→ 5 - FU的转化。结果 :PCR法扩增出全长 YCD基因 ,经测序证实序列正确 ;构建了 MSCV - YCD- IRES- EGFP逆转录病毒载体 ,载体转染包装细胞Ф NX- A,获得滴度为 3.5× 10 6 CFU / m l的逆转录病毒 ;病毒转染 K5 6 2 B,转染率为 30 % ,经筛选获得转基因阳性克隆 YCD- K 5 6 2 B,RT- PCR检测显示 YCD- K5 6 2 B有 YCD基因的 m RNA表达 ,其细胞裂解产物可使 5 - FC转化为 5 - FU,表明其有 CD酶活性 ;MTT法检测低浓度 5 - FC(15μmol/ L )作用 96 h对 YCD- K5 6 2 B有明显的杀伤效应。 结论 :YCD/ 5 - FC系统对转基因肿瘤细胞有明显的杀伤效应。

Objective:To clone the yeast cytosine deaminase(YCD) gene and to elucidate the killing efficacy of yeast cytosine deaminase / 5 - fluorocytosine gene therapy system on gene- transfered cells. Methods:YCD gene was cloned and YCD expression retroviral vector was constructed,the vector was transfered into packaging cell line and high- titer virus was abtained. Then the tum origenic leukemia cell line K5 6 2 B was infected,selected and evaluated. Finally,the killing efficacy of 5 - FC on gene- transfered cell clone observed,and the 5 - FC to 5 - FU conversion of YCD- K5 6 2 B cell lysate was estim ated. Results:YCD gene was cloned and the sequence was confirm ed. A YCD expression retroviral vector,MSCV- YCD- IRES- EGFP was constructed. Transfering it into packaging cell lineФ NX- A ,high- titer (3.5× 10 6 CFU / ml) virus was obtained. The virus were used to infect tumorigenic leukem ia cell line,K5 6 2 B,and the infecting rate was quite high (30 % ) . A gene- transfered cell clone,YCD- K5 6 2 B,was selected,and YCD gene m RNA expression was detected in it.YCD- K5 6 2 B cell lysate demonstrated CD enzyme activity,and it could deaminase 5 - FC to 5 - FU .MTT assay showed 5 - FC could kill YCD- K5 6 2 B cell in 96 h even at lower concentration(15μmol/ L ) . Conclusion:YCD/ 5 - FC suicide gene therapy system has a significantkilling efficacy on gene- transfered cells. [