You Position: Home > Paper
shRNA-mediated construction of RNAi expression plasmid and its inhibitory effect on MDR1 expression in hepatocellular carcinoma cell SMMC-7721/R
( views:267, downloads:4 )
- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 18
- DOI:
- 10.3321/j.issn:1000-2790.2007.18.003
- Key Word:
- RNA干扰;抗药性,多药;短发夹RNA;重组,遗传;质粒;基因表达
Abstract： 目的:重组构建抑制多药耐药MDR1基因表达的shRNA真核表达载体,研究其对肝癌耐药细胞MDR1基因表达的抑制作用,以及对P-糖蛋白(P-gp)表达和功能的抑制作用.方法:根据MDR1基因序列,设计两段21个碱基的MDR1特异性靶序列作为shRNA目标序列(sh-MDR1-1,sh-MDR1-2),重组构建psh-MDR1表达质粒.采用脂质体介导转染肝癌耐药细胞SMMC-7721/R,用MTT法测定细胞对化疗药物阿霉素(ADM)的敏感性,流式细胞仪检测细胞膜表面P-糖蛋白(P-gp)表达和细胞内Rhdaming123(Rh123)的潴留.结果:PCR和DNA测序证实了psh-MDR1-1和psh-MDR1-2重组质粒的成功构建,均能有效地抑制细胞P-gp表达;转染细胞后均能够一定程度地恢复耐药细胞对化疗药物ADM敏感性,P-gp表达水平明显降低,细胞内的Rh123稳态积累量均明显增高;第1条序列更能有效的抑制MDR1基因表达.结论:体外完成shRNA RNAi载体的构建,能有效地逆转肝癌耐药细胞MDR1基因过度表达,抑制P-gp介导的多药耐药.从DNA载体产生的shRNA在肝癌耐药细胞内能诱导RNAi,能够序列特异性地抑制MDR1基因表达.
- [1]王玮,岑建农,李建勇,等.急性白血病中MDR1 MRP和bcl-2基因表达及其临床意义[J].2003,(7).
- [2]刘家云,李庆霞,黄红艳,等.瞬时转染和稳定转染对RNAi抑制乙型肝炎病毒S基因表达的影响[J].2005,(11).
- [3]亓同钢,汪运山.RNA干涉在肿瘤研究中的应用进展[J].2003,(12).
- [4]Roots I,Pahkla R,Oselin K,etc.MDR1 polymorphisms G2677T in exon 21 and C3435T in exon 26 fail to affect rhodamine 123 efflux in peripheral blood lymphocytes.[J].2003,17(4).
- [5]Shi Y,Forrester WC,Sui G,etc.A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.[J].2002,99(8).
- [6]Thomas, Tuschl.Expanding small RNA interference.[J].2002,20(5).
- [7]Lowenadler B,Rhedin M,Langguth P,etc.Selective downregulation of the MDR1 gene product in Caco-2 cells by stable transfection to prove its relevance in secretory drug transport.[J].2005,2(1).
- [8]Gregory J, Hannon.RNA interference.[J].2002,418(6894).