核糖核酸干扰抑制P-糖蛋白在大鼠耐药星形胶质细胞中过度表达  

RNA interference inhibiting permeability glycoprotein over-expression in pharmaco-resistance rat astrocytes model

作　　者：陈蕾[1] 田林郁[1] 杨天华[1] 周东[1] 

机构地区：[1]四川大学华西医院神经内科,成都610041

出　　处：《中华神经科杂志》2008年第10期699-703,共5页Chinese Journal of Neurology

基　　金：国家自然科学基金资助项目（0040205401094）

摘　　要：目的利用腺病毒载体介导的短发夹RNA（shRNA）抑制大鼠多药耐药基因mdr1b及其编码的P-糖蛋白（Pgp）的表达，探讨其改善癫痫耐药现象的可行性。方法通过同源重组方法构建腺病毒，表达针对mdr1b的shRNA，感染马桑内酯诱导的耐药星形胶质细胞模型。实时定量逆转录-聚合酶链反应（RT—PCR）和Westernblot法分别检测感染5d内细胞mdr1b和Pgp的表达，流式细胞术检测罗丹明泵出率。结果得到6X10^10pfu／ml滴度的重组腺病毒，转染星形胶质细胞后，细胞mdr1b和Pgp表达明显被抑制，干扰率达94％（P〈0．01）。Ad5-EGFP—shRNA1-U6感染组细胞罗丹明泵出率15．8％，明显低于对照组（56．2％，F=127．5，P〈0．05）。结论成功构建针对大鼠mdr1b的RNAi腺病毒载体，并在体外证实其对大鼠mdr1b表达的高效抑制作用，为进一步探索难治性癫痫的基因治疗奠定基础。Objective To study the effect of adenoviral-delivered short hairpin RNA （shRNA） target against permeability glycoprotein （Pgp） as a new drug in anti-epileptic drug resistance epilepsy treatment and to evaluate its efficiency. Methods MDR Sprague-Dawley （ SD ） rat astrocyte model was induced by Coriaria Lactone （CL）, mainly over-expressing mdr1b. To reverse the drug resistance, astrocytes were treated with constructed replication deficient adenovirus Ad5-EGFP-shRNA1-U6 delivering short hairpin （shRNA） target agianst mdr1b gene. Total RNA and protein were extracted from the infected cells, mdr1b level was detected by Quantitative Real-time PCR whereas Pgp by Western blot, Rhodamine123 （ Rho123 ） efflux ratio by Flow Cytometry. Results Ad5-EGFP-shRNA1-U6 was succesfully constucted with high virus titer of 6 × 10^10 pfu/ml. The interference efficency of AdS-EGFP-shRNA1-U6 agianst mdrl b in rat astrocyte model was about 94%. The Rho123 efflux ratio was about 15.8%, significiently lower than control group which was 56. 2% （F = 127.5, P 〈 0. 05 ）. Conclusions Pgp over-expression has been successfully suppressed and MDR has been reversed, which may provide a promising approach for refractory epilepsy remedy.

关 键 词：腺病毒科 RNA 小分子干扰 P糖蛋白 星形细胞 转染 

分 类 号：R686[医药卫生—骨科学]