CAJ | 학술논문

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,探讨其改善癫疴耐药现象的可行性.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Western blot法分别检测感染5 d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6×1010pfu/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).Ads-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癎的基因治疗奠定基础.
목적 이용선병독재체개도적단발협RNA(shRNA)억제대서다약내약기인mdr1b급기편마적P-당단백(Pgp)적표체,탐토기개선전아내약현상적가행성.방법 통과동원중조방법구건선병독,표체침대mdr1b적shRNA,감염마상내지유도적내약성형효질세포모형.실시정량역전록-취합매련반응(RT-PCR)화Western blot법분별검측감염5 d내세포mdr1b화Pgp적표체,류식세포술검측라단명빙출솔.결과 득도6×1010pfu/ml적도적중조선병독,전염성형효질세포후,세포mdr1b화Pgp표체명현피억제,간우솔체94%(P<0.01).Ads-EGFP-shRNA1-U6감염조세포라단명빙출솔15.8%,명현저우대조조(56.2%,F=127.5,P<0.05).결론 성공구건침대대서mdr1b적RNAi선병독재체,병재체외증실기대대서mdr1b표체적고효억제작용,위진일보탐색난치성전간적기인치료전정기출.
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 estrocyte model was induced by Coriaria Lactone (CL), mainly over-expressing mdrlb. To reverse the drug resistance, astrecytes were treated with constructed replication deficient adencvirus AdS-EGFP-shRNAI-U6 delivering short hairpin (shRNA) target agianst mdrlb gene. Total RNA and protein were extracted from the infected cells, mdr1 b level was detected by Quantitative Real-time PCR whereas Pgp by Western blot, Rhodamine123 (Rho123) efflux ratio by Flow Cytometry. Results AdS-EGFP-shRNA1-U6 was succesfully constucted with high virus titer of 6×1010 pfu/ml. The interference efficency of AdS-EGFP-shRNA1-U6 agianst mdrlb in rat astrecyte model was about 94%. The Rho123 efllux 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 premising approach for refractory epilepsy remedy.