摘要
目的 研究抗肿瘤细胞多药抗性 (MDR)核酶的生物学活性及其稳定性。方法 构建表达抗MDR1核酶的逆转录病毒载体 (N2A +tRNAmeti 196MDR1 Rz、N2A +tRNAmeti 196MDR1 sRz、N2A +tRNAmeti 1iMDR1 sRz)和合成了裸核酶cDNA。利用体外转录反应合成 5种anti MDR1核酶 (tRNA 196MDR1 Rz、tRNA 196MDR1 sRz、tRNA iMDR1 sRz、196MDR1 sRz和 196MDR1 Rz)及相应的底物A和底物B。在无细胞体系中检测了各种核酶的切割活性和它们对核酸酶降解的稳定性。结果 anti MDR1核酶均能有效发挥切割作用。其中嵌合型核酶的活性高于裸核酶 ,而且经过stem Ⅱ碱基修饰的核酶催化活性较高。核酶的稳定性依次是tRNA 196MDR1 sRz(嵌合型 ) >tRNA 196MDR1 Rz =196MDR1 sRz(裸核酶 )。在对照组 ,突变的tRNA mut iMDR1 sRz没有切割活性。结论 核酶分子经过stem
Objective Development of multidrug resistance(MDR) is the major obstacle to successful cancer chemotherapy. One strategy to block the P glycoprotein(P gp) mediated MDR is to use a ribozyme (Rz) target against MDR1 mRNA.Methods Three kinds of anti MDR1 chimeric hammerhead ribozymes, the first one cleaving codon 196 of MDR1 mRNA (196MDR1 Rz), the second one, a stem Ⅱ base modified (U 9→G 9,U 13 →A 13 ,G 14 →A 14 ,A 18 →C 18 ) Rz against codon 196 (196MDR1 sRz) and the third one, the stem Ⅱ base modified Rz directed against the -6～ 4 GUC sequence of the translation initiation site of the MDR1 mRNA (iMDR1 sRz), were synthesized based on the cloned retroviral constructs: N2A+ tRNA met i 196MDR1 Rz, N2A+ tRNA met i 196MDR1 sRz, N2A+ tRNA met i iMDR1 sRz.Results In a cell free system, the chimeric tRNA sRz molecules were more stable and had more efficient catalytic activities than the corresponding naked Rz molecules. The stem II base modified Rz molecules were also more stable and efficient in catalytic activities than the unmodified ones. In control, the disabled tRNA mut iMDR1 sRz had no cleavage activity. Conclusion Base modification in the Rz′s stem Ⅱ of ribozyme structure and the development of chimeric tRNA ribozyme molecules are able to enhance the cleavage efficacy.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2000年第3期184-188,共5页
Chinese Journal of Oncology
