大鼠坐骨神经损伤后背根神经节microRNA的表达变化及其功能的初步研究
发布时间：2019-01-02 20:35
【摘要】：目的研究大鼠坐骨神经损伤后背根神经节(Dorsal root ganglion, DRG) microRNA(miRNA)的表达变化及其功能。 方法选用健康成年雄性60-90日龄Spragu-Dawley(SD)大鼠,建立右侧坐骨神经切断损伤模型,3天后分别提取损伤侧和正常侧(对照)与坐骨神经相连的腰4-5(L4-5)DRG的总RNA,运用miRNA芯片技术筛选损伤后表达量出现差异的miRNA;进一步采用实时定量PCR(Quantitative real-time polymerase chain reaction,qRT-PCR)和原位杂交方法对芯片差异表达miRNA进行验证。运用生物信息学软件,根据芯片及qRT-PCR结果对筛选的miRNA进行靶基因预测；根据预测结果,构建表达载体运用荧光素酶报告基因系统对候选miRNA的靶基因进行确认。最后,在培养的大鼠DRG感觉神经元,转染筛选获得的目的miRNA,运用(3-tubulin Ⅲ免疫荧光组织化学染色观察其对神经元神经突长度的影响、Western blotting观察其对Robo2蛋白表达的影响研究其功能。 结果MiRNA芯片结果显示坐骨神经切断后,DRG内有21种miRNA的表达出现下调。qRT-PCR及原位杂交结果均显示miR-144、 miR-145和miR-214的表达均下调,与芯片结果一致。生物信息学预测显示Robo2和srGAP1的3’-非翻译区(3'-untranslated regions,3'-UTR)存在miR-144、miR-145和miR-214的结合位点,荧光素酶报告基因系统结果显示miR-145和miR-214能分别抑制Robo2和srGAP1的表达。Western blotting结果显示培养的DRG感觉神经元经转染miR-145后,Robo2的蛋白表达明显下调,确认其是miR-145的靶基因,并且神经突长度缩短,证实miR-145调控DRG感觉神经元的神经突生长。 结论坐骨神经损伤后DRG神经元miRNA发生差异表达。其中,miR-145和miR-214表达明显下调,miR-145抑制神经突的生长,其机制可能通过下调Robo2表达；miR-214可能调控srGAP1的表达。miRNA可能调控Slit-Robo-srGAP通路分子参与周围神经再生过程。
[Abstract]:Objective to study the expression and function of (Dorsal root ganglion, DRG) microRNA (miRNA) in dorsal root ganglion (DRG) after sciatic nerve injury in rats. Methods healthy adult male Spragu-Dawley (SD) rats aged 60-90 days were selected to establish the right sciatic nerve transection injury model. Three days later, the total RNA, of the injured side and the normal side (control) and the lumbar 4-5 (L4-5) DRG connected with the sciatic nerve were extracted respectively. MiRNA chip technique was used to screen the miRNA; with different expression levels after the injury. Furthermore, real-time quantitative PCR (Quantitative real-time polymerase chain reaction,qRT-PCR and in-situ hybridization were used to verify the differential expression miRNA. Bioinformatics software was used to predict the target genes of selected miRNA according to the results of microarray and qRT-PCR, and the expression vector was constructed to identify the target genes of candidate miRNA by luciferase reporter gene system. Finally, in cultured rat DRG sensory neurons, transfection and screening were carried out to investigate the effect of miRNA, (3-tubulin 鈪，
本文编号：2398957
[Abstract]:Objective to study the expression and function of (Dorsal root ganglion, DRG) microRNA (miRNA) in dorsal root ganglion (DRG) after sciatic nerve injury in rats. Methods healthy adult male Spragu-Dawley (SD) rats aged 60-90 days were selected to establish the right sciatic nerve transection injury model. Three days later, the total RNA, of the injured side and the normal side (control) and the lumbar 4-5 (L4-5) DRG connected with the sciatic nerve were extracted respectively. MiRNA chip technique was used to screen the miRNA; with different expression levels after the injury. Furthermore, real-time quantitative PCR (Quantitative real-time polymerase chain reaction,qRT-PCR and in-situ hybridization were used to verify the differential expression miRNA. Bioinformatics software was used to predict the target genes of selected miRNA according to the results of microarray and qRT-PCR, and the expression vector was constructed to identify the target genes of candidate miRNA by luciferase reporter gene system. Finally, in cultured rat DRG sensory neurons, transfection and screening were carried out to investigate the effect of miRNA, (3-tubulin 鈪，
本文编号：2398957
本文链接：https://www.wllwen.com/xiyixuelunwen/2398957.html
最近更新
教材专著
