摘要
本研究应用载体介导的RNA干扰(RNA interference,RNAi)技术特异地干扰孤儿核受体1(nuclear related factor1,Nurr1)基因的表达,以建立稳定的RNAi技术及用于下一步的Nurr1基因治疗研究。我们筛选了四对Nurr1基因的特异性序列shRNA(short hairpin RNA),构建相应的载体(psilencerTM3.1-H1neo shRNA1,2,3,4)。分别将这4种质粒瞬时转染入已转染了逆转录病毒质粒pLNCX2-Nurr1并稳定表达Nurr1基因的PT67细胞中。在转染后24、48和72h分别采用免疫荧光、免疫组化、免疫印迹以及Real-time PCR的方法检测细胞中Nurrl蛋白及基因的表达水平。结果显示,转染psilencerTM3.1-H1neo shRNA2、3号24h和48h后,经免疫荧光和免疫组化检测,Nurr1在PT67细胞中表达显著降低;免疫印迹结果也显示,Nurr1蛋白量明显低于阴性对照组。psilencerTM3.1-H1neo shRNA1、4对Nurr1蛋白表达没有明显影响。Real-time PCR相对定量结果也同样证实2、3号转染后Nurr1基因表达的干扰效果明显,而1、4号结果相对较弱。通过本实验,我们筛选出了两段Nurr1特异性siRNA序列,且干扰效果在转染后24h至48h时效果最为明显。
In order to establish stable RNA interference （RNAi） technique and use this technique for further gene therapy, we used vector-medlated RNAi technique in vitro to suppress the expression of nuclear related factor1 （ Nurr1 ） gene. Four different sbRNA sequences specific to Nurr1 gene were selected and their eukaryotic expression vectors （ psilencer^TM 3.1 -H1 neo shRNA 1,2, 3,4 ） were constructed. The vectors with different Nurr1 shRNA were transfected into PT67 cells in which the Nurr1 gene was delivered by retroviral vector pLNCX2-Nurr1. At 24, 48 and 72 h after transfection, the expression level of Nurr1 was detected by immunochemistry, immunofluorescence assay, Western blotting and Real-time PCR. The results showed that the expression of Nurr1 was significantly inhibited in PT67 cells by the psilencer^TM3. 1-H1neo shRNA 2 and the psilencer^TM 3, 1-Hlneo shRNA 3 at 24 h and 48 h after transfection revealed by immunochemistry and immunofluorescence methods as well as Western blotting. The psilencer^TM 3. 1-Hlneo shRNA 1 and the psilencer^TM3. 1- H1 neo shRNA 4 did not affect the expression level of Nurr1. Real-time PCR confirmed above mentioned results. The present study screened two specific shRNA sequences to Nurr1 gene which could efficiently suppress the expression of Nurr1 gene, and the effect was apparent at 24 -48 h after transfection.
出处
《神经解剖学杂志》
CAS
CSCD
北大核心
2007年第2期121-126,共6页
Chinese Journal of Neuroanatomy
基金
首都医科大学校基金
教育部留学人员回国基金(2004-527)
北京市优秀人员专项基金资助项目(20042D0501801)
教育部博士点基金(20040025007)资助项目
