【摘要】：目的研究大鼠坐骨神經(jīng)損傷后背根神經(jīng)節(jié)(Dorsal root ganglion, DRG) microRNA(miRNA)的表達變化及其功能。 方法選用健康成年雄性60-90日齡Spragu-Dawley(SD)大鼠,建立右側(cè)坐骨神經(jīng)切斷損傷模型,3天后分別提取損傷側(cè)和正常側(cè)(對照)與坐骨神經(jīng)相連的腰4-5(L4-5)DRG的總RNA,運用miRNA芯片技術(shù)篩選損傷后表達量出現(xiàn)差異的miRNA;進一步采用實時定量PCR(Quantitative real-time polymerase chain reaction,qRT-PCR)和原位雜交方法對芯片差異表達miRNA進行驗證。運用生物信息學軟件,根據(jù)芯片及qRT-PCR結(jié)果對篩選的miRNA進行靶基因預(yù)測；根據(jù)預(yù)測結(jié)果,構(gòu)建表達載體運用熒光素酶報告基因系統(tǒng)對候選miRNA的靶基因進行確認。最后,在培養(yǎng)的大鼠DRG感覺神經(jīng)元,轉(zhuǎn)染篩選獲得的目的miRNA,運用(3-tubulin Ⅲ免疫熒光組織化學染色觀察其對神經(jīng)元神經(jīng)突長度的影響、Western blotting觀察其對Robo2蛋白表達的影響研究其功能。 結(jié)果MiRNA芯片結(jié)果顯示坐骨神經(jīng)切斷后,DRG內(nèi)有21種miRNA的表達出現(xiàn)下調(diào)。qRT-PCR及原位雜交結(jié)果均顯示miR-144、 miR-145和miR-214的表達均下調(diào),與芯片結(jié)果一致。生物信息學預(yù)測顯示Robo2和srGAP1的3’-非翻譯區(qū)(3'-untranslated regions,3'-UTR)存在miR-144、miR-145和miR-214的結(jié)合位點,熒光素酶報告基因系統(tǒng)結(jié)果顯示miR-145和miR-214能分別抑制Robo2和srGAP1的表達。Western blotting結(jié)果顯示培養(yǎng)的DRG感覺神經(jīng)元經(jīng)轉(zhuǎn)染miR-145后,Robo2的蛋白表達明顯下調(diào),確認其是miR-145的靶基因,并且神經(jīng)突長度縮短,證實miR-145調(diào)控DRG感覺神經(jīng)元的神經(jīng)突生長。 結(jié)論坐骨神經(jīng)損傷后DRG神經(jīng)元miRNA發(fā)生差異表達。其中,miR-145和miR-214表達明顯下調(diào),miR-145抑制神經(jīng)突的生長,其機制可能通過下調(diào)Robo2表達；miR-214可能調(diào)控srGAP1的表達。miRNA可能調(diào)控Slit-Robo-srGAP通路分子參與周圍神經(jīng)再生過程。
[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，

本文編號：2398957