核酶体外切割肝癌多药耐药基因的研究  

Cleavage of human MDR1 mRNA by hammerhead ribozymes

作　　者：刘顺英[1] 孙宁[1] 

机构地区：[1]南京铁道医学院附属医院消化科,210009

出　　处：《江苏医药》2000年第1期24-26,共3页Jiangsu Medical Journal

摘　　要：目的　利用核酶技术切割肿瘤多药耐药基因 (MDR1 ) ,逆转肿瘤抗药性。方法　针对人类MDR1mRNA第VⅡ外显子附近第 1 79及 1 96密码子的GUC序列 ,按照“锤头结构”模型设计、合成了两个核酶 1 79MDR1Ribozyme(1 79RZ)和 1 96MDR1Ribozyme(1 96RZ) ,并定向克隆入载体PGEM 3Zf(+)中。从肝癌多药耐药细胞株SMMC - 772 1 /DOX中提取MDR1mRNA ,RT -PCR法扩增出约 2 6 9bp大小的特异性cDNA片段 ,采用SmaRT法克隆入载体PGEM - 3Zf(+)中。在体外转录成RNA。结果　通过测序证实重组质粒插入片断正确 ,在生理条件下 ,两个核酶均能定点切割MDR1mRNA成两段特定大小的片段。核酶的切割效率与作用时间、核酶与底物的比率、Mg2 + 浓度有关 ,1 96RZ的切割活性高于 1 79RZ。结论　核酶技术逆转肿瘤抗药性具有现实可能性。Objective To reverse P glycoprotein mediated drug resistance specifically. Method we designed two hammerhead ribozymes which can cleave the GUC sequence in codon 179 and 196 of MDR1(PGY1) mRNA.A target MDR1 mRNA, around the codon 185 which codes for an amino acid residue possibly influencing the drug binding function of the P gp,was created by a reverse transcription polymerase chain reaction using a SMMC 7721 human hepatic carcinoma cell line resistant to Doxorubicin(SMMC 7721/DOX),which displayed MDE1 overexpression.The MDR1 cDNA was then cloned by SmaⅠ Re ligation Technique(sma RT). Results In a cellfree system,both ribozymes cleaved a target piece of MDR1 mRNA into two fragments at the specific site at physiological pH and temperature.The cleavage reaction was dependent on ribozyme:substrate ratio, incubation time and Mg 2+ ion.The 196 MDR1 ribozyme was more active than the 179 MDR1 ribozyme. Conclusion The results show that ribozymes may inactivate MDR1 mRNA and revert the multidrug resistance phenotype because of their simple structure,site specific cleavage activity and catalytic potential.

关 键 词：多药耐药基因 核酶 基因克隆 肝癌 RT-PCR 

分 类 号：R735.7[医药卫生—肿瘤]