CAJ | 학술논문

目的:构建大肠埃希菌胞嘧啶脱氨酶(E. coli cytosine deaminase,EC-CD)基因突变体D314A(即EC-CD基因开放阅读框第314位氨基酸由天冬氨酸突变为丙氨酸)并研究其抗肿瘤作用.方法:构建含EC-CD基因的真核表达质粒pcDNA3.1-CD~(wt),应用点突变技术将pcDNA3.1-CD中EC-CD基因开放阅读框第314位氨基酸的碱基由A突变为C,即构成pcDNA3.1-CD~(D314A).用Lipofectamine ~(TM) 2000将EC-CD基因或D314A基因转入人结肠癌细胞系LoVo细胞,以G418筛选出稳定表达的阳性克隆LoVo-CD~(wt)及LoVo-CD~(D314A).应用MTT法检测EC-CD基因及D314A基因对LoVo细胞的直接杀伤作用及旁观者效应.结果:成功构建EC-CD基因突变体D314A并经测序确认.LoVo细胞转染EC-CD基因及D314A基因后均能表达相应的mRNA,Lovo-CD~(D314A)细胞对5-FC的IC_(50)为(85.13±0.60)mmol/L,显著低于LoVo-CD~(wt)细胞的(689.76±0.45)μmoL/L,(P=0.000);在旁观者试验中,当LoVo-CD~(wt)细胞和LoVo-CD~(D314A)细胞比例均为30%时,细胞存活率分别为(48.5±0.49)%与(17.3±0.40)%(P=0.000).结论:EC-CD基因突变体D314A的抗LoVo细胞作用显著强于野生型EC-CD基因,有望成为新的肿瘤基因治疗候选基因.
목적:구건대장애희균포밀정탈안매(E. coli cytosine deaminase,EC-CD)기인돌변체D314A(즉EC-CD기인개방열독광제314위안기산유천동안산돌변위병안산)병연구기항종류작용.방법:구건함EC-CD기인적진핵표체질립pcDNA3.1-CD~(wt),응용점돌변기술장pcDNA3.1-CD중EC-CD기인개방열독광제314위안기산적감기유A돌변위C,즉구성pcDNA3.1-CD~(D314A).용Lipofectamine ~(TM) 2000장EC-CD기인혹D314A기인전입인결장암세포계LoVo세포,이G418사선출은정표체적양성극륭LoVo-CD~(wt)급LoVo-CD~(D314A).응용MTT법검측EC-CD기인급D314A기인대LoVo세포적직접살상작용급방관자효응.결과:성공구건EC-CD기인돌변체D314A병경측서학인.LoVo세포전염EC-CD기인급D314A기인후균능표체상응적mRNA,Lovo-CD~(D314A)세포대5-FC적IC_(50)위(85.13±0.60)mmol/L,현저저우LoVo-CD~(wt)세포적(689.76±0.45)μmoL/L,(P=0.000);재방관자시험중,당LoVo-CD~(wt)세포화LoVo-CD~(D314A)세포비례균위30%시,세포존활솔분별위(48.5±0.49)%여(17.3±0.40)%(P=0.000).결론:EC-CD기인돌변체D314A적항LoVo세포작용현저강우야생형EC-CD기인,유망성위신적종류기인치료후선기인.
Objective:To construct a mutant D314A of Escherichia coli cytosine deaminase (EC-CD, substitution of an alanine (A) for the aspartic acid (D) at position 314 of cytosine deaminase) and investigate its antitumor effect. Methods: Eukaryotic expression plasmid containing EC-CD gene (pcDNA3.1-CD~(wt)) was constructed, and the mutant pcDNA3.1-CD~(D314A) plasmid, with aspartic acid (D) at position 314 of EC-CD gene substituted by alanine (A) (EC-CD~(D314A)), was established by site-directed mutation. EC-CD~(wt) and EC-CD~(D314A) were transfected into human colon cancer cell line LoVo via Lipofectamine~(tm) 2000, and positive LoVo-CD~(wt) and LoVo-CD~(D314A) cells stably expressing corresponding genes were selected by G418. The cytotoxicity and bystander effects of EC-CD and EC-CD~(D314A) genes on LoVo cells were e-valuated by MTT assay. Results: The mutant D314A was confirmed by sequence analysis. EC-CD and EC-CD~(D314A) mRNA were expressed after transfected into LoVo cells. The IC_(50) of Lovo-CD~(D314A) cells was (85.13±0.60) mmol/L, which was significantly lower than that of LoVo-CD~(wt) cells ([689.76±0.45] μmol/L, P=0.000). Bystander effect assay showed that, when at the ratio of 30%, the survival rates of LoVo-CD~(wt) cells and Lovo-CD~(D314A) cells were (48.5±0.49)% and (17.3±0.40) % (P = 0.000), respectively. Conclusion: Mutatant EC-CD gene (EC-CD~(D314A)) has a significantly in-creased antitumor effect on LoVo cells compared with wild type EG-CD gene, and it may become a new candidate gene for tumor gene therapy.