永生化小鼠小胶质细胞P_2X_4siRNA靶点的筛选

Screening of P_2X_4siRNA on Immortalized Mice Microglia

目的筛选永生化小鼠小胶质细胞特异性P2X4siRNA。方法转染FAM-siRNA16h后,激光扫描共聚焦显微镜检测永生化小鼠小胶质细胞转染效率;将3条特异性siRNA经lipofectamine2000介导转染永生化小鼠小胶质细胞;72h后半定量RT-PCR观察干扰前后P2X4基因mRNA水平表达,比较对应不同位点的三对siRNA片段对P2X4基因的干扰效果,分析RNA干扰的特异性,从中筛选出特异性最高的一条siRNA;转染该序列72h后Western blot检测P2X4基因蛋白表达。结果激光扫描共聚焦显微镜显示转染效率达80%;三对siRNA片段中siRNA-F1的沉默效率最高,最大干扰效率达72.2%;Western blot显示转染siRNA-F1后P2X4基因蛋白表达下调。结论siRNA-F1可有效下调永生化小鼠小胶质细胞中P2X4基因mRNA表达水平,并且能明显下调P2X4蛋白表达。

Objective To screen the best P2X4siRNA on immortalized mice microglia. Methods FAM-siRNA was transfected into immortalized mice microglia, and transfection efficiency was detected by laser scanning confocal microscope （LSCM） after 16 h. Immortalized mice microglia was transfected with 3 siRNAs （different loci） of P2 X4 by lipofectmaine 2000. The levels of P2 X4 mRNA expression before and after transfection were detected by semi-quantitative reverse transcription polymerase chain reaction （RT-PCR）. The interfering effect between the 3 siRNAs and the specificity for interference were compared 72 h after transfection, and the best siRNA was screened. The levels of P2 X4 protein expression before and after transfection were detected by Western blot. Results The transfection efficiency was 80% detected by LSCM. The levels of P2 X4 mRNA in immortalized mice microglia were inhibited by the specific siRNA-F1 , and the most apparent interfering efficiency was 72.2M 72 h after transfection. Western blot indicated that the expression of P2 X4 protein was downregulated in immortalized mice microglia, Conclusion P2 X4 siRNA can downregulate the level of P2 X4 mRNA in immortalized mice microglia. And P2 X4 protein expression is also downregulated by specific siRNAs.