脱氧核酶对肝癌细胞AFPmRNA表达的影响  

Effects of deoxyribozyme on AFPmRNA expression in liver tumor cell

作　　者：初秋[1] 罗速[1] 杨春玫[1] 

机构地区：[1]北华大学基础医学院,吉林吉林市132013

出　　处：《中国实验诊断学》2007年第11期1464-1466,共3页Chinese Journal of Laboratory Diagnosis

基　　金：吉林省科技发展计划项目(200505249)

摘　　要：目的本研究针对AFPmRNA(甲胎蛋白mRNA)设计脱氧核酶,观察其切割AFPmRNA的有效性,探索脱氧核酶在肝癌基因治疗的可能性。方法体外转录AFPmRNA底物,设计并合成DZ(脱氧核酶)、DZs(硫代修饰脱氧核酶,序列同DZ,但两侧各有两个脱氧核苷酸进行了硫代修饰)、ASO(反义寡聚脱氧核苷酸)、无关DZ对照。体外切割AF-PmRNA;经转染试剂将脱氧核酶转染入肝癌细胞HepG2,利用RT-PCR法检测AFPmRNA水平的变化;免疫细胞化学及图像分析系统检测AFP蛋白质表达情况;MTT法初步估计肝癌细胞的生长效应。结果DZ与DZs在体外均可切割AFPmRNA底物;经转染DZ、DZs的细胞均下降AFPmRNA的水平,而且DZs的作用更强;经转染DZ与DZs的细胞均下降AFP的表达,ASO也略下降AFP的表达;DZ、DZs有抑制细胞生长的作用。结论本实验设计的脱氧核酶能在细胞外有效切割AFPmRNA;在细胞内也能切割AFPmRNA、抑制AFP的表达、抑制肝癌细胞的生长,为肝癌的基因治疗提供可能性。Objective To design deoxyribozyme(DZ) for AFPmRNA(alpha fetoprotein mRNA),and to detect the catalytic activities in AFPmRNA.Explore the possibility for AFP gene therpy by using DZ.Methods The AFPmRNA fragments were transcripited in vitro.To design and synthesize DZ,DZs,ASO,no connection with DZ.DZ etc.were transfected into cells,RT-PCR was performed for evaluating the inhibitory activity of DZ etc.on AFPmRNA expression,immunocytochemical methods and Image were used to detect the AFP protein expression on cells,MTT assay were performed to detect the cell activity of cells.Results DZ and DZs were able to cut AFPmRNA in cells free system.DZ and DZs were capable of decreasing the AFPmRNA expression.DZ and DZs can inhibit the AFP protein expression and the growth of the liver cell obviously.Conclusion Under the proper ion concentration,temperature and pH conditions,in free system DZ and DZs targeting AFP mRNA can cleavge the substrate RNA specifically and efficiently,the expression of AFPmRNA was inhibited by DZ and DZs in cell system,synthesis of AFP and growth were inhibited by DZ and DZs.

关 键 词：脱氧核酶 AFPMRNA AFP RT-PCR 

分 类 号：R735.7[医药卫生—肿瘤]