DOX-GA3对βG转染人肾癌GRC-1细胞的体外杀伤作用

Cytotoxicity of DOX-GA3 to β-glucuronidase cDNA transfected human renal carcinoma GRC-1 cells in vitro

目的建立高表达β-葡萄糖醛酸苷酶(βG)基因的肾癌模型,并观察βG前体药葡萄糖醛酸化阿霉素(DOX-GA3)对βG转染人肾癌GRC-1细胞的体外杀伤作用.方法利用阳离子脂质体将已构建的真核表达载体pcDNA3.1(+)转入肾癌细胞株GRC-1,用基因组PCR扩增,免疫荧光,免疫组化检测鉴定其转录和表达水平,分别用MTT及细胞计数法检测βG阳性细胞对DOX-GA3的体外敏感性和旁观者效应.结果经G418筛选后的阳性细胞克隆,在DNA和蛋白水平证实了转染细胞内有βG基因高表达,100μmol/L DOX-GA3作用于亲本细胞3d,生存率为82%,但可杀死全部转染细胞,IC50GRC-1/βG为1.24μmol/L,而IC50GRC-1为100μmol/L以上,将转染细胞和亲本细胞以不同比例混合时,转染细胞占20%,有80%的细胞被杀死.结论建立了稳定的βG基因高表达的肾癌模型.DOX-GA3对转染细胞有明显的杀伤作用,随时间延长和剂量增加作用增强,并显示较强的旁观者效应.

AIM： To establish a renal carcinoma model with high expression of β-glucuronidase （βG） and to observe the killing effect of βG-prodrug N-[4-Doxorubicin-N-carbonyl （ oxymethyl ） phehyl ] -O-beta-glucuronyl carbamate （ DOX- GA3） on βG cDNA transfected human renal carcinoma GRC-1 cells in vitro. METHODS： A eukaryotic expression vector pcDNA3.1 （ ＋ ）-βG was transferred into renal carcinoma GRC-1 cells with lipofection reagent and the βG vector-producer cell line GRC-1βG was isolated in 1000 mg/L neomycin medium. It was confirmed by genomic PCR, immunofluorescency and immunohistochemistry. The cytotoxicity and bystander effect were estimated by MTT and trypan blue exclusion test, RESULTS： βG gene was stably integrated into the genomic DNA of the GRC-1 cell and got a high expression. The DOX-GA3, with the concentration of 100 μmol/L for 3 d, had little effect on parental cells, with a survival rate of 82%. However, it killed all the transfected cells. When IC50 GRC-1βG was 1. 24 lamol/L, IC50 GRC- 1 was more than more than 100 μmol/L, and the GRC-1βG cells were mixed with parental cells with various proportions, the GRC-1βG cells accounted for 20%, 80% of the mixed cells were killed. CONCLUSION： We have successfully established the renal carcinoma model with high expression of βG gene. DOX-GA3 has obvious cell killing effect on the GRC-1βG, which is strengthened with the rising concentration and the prolonging time, and potent bystander effect.