【摘要】：目的周圍神經(jīng)損傷后再生是一個極其復(fù)雜的過程,損傷的神經(jīng)組織中有多種基因和蛋白的表達發(fā)生改變。microRNAs(miRNAs)作為一類小的非編碼RNA在基因轉(zhuǎn)錄后水平發(fā)揮調(diào)控作用,其發(fā)現(xiàn)為研究神經(jīng)損傷與再生過程中的分子機制提供了一個新的作用模式。施萬細胞是周圍神經(jīng)系統(tǒng)主要的膠質(zhì)細胞,能改善周圍神經(jīng)微環(huán)境,在周圍神經(jīng)損傷后的變性和再生中有著非常重要的作用。本課題旨在探討坐骨神經(jīng)損傷與再生過程中miR-9對施萬細胞功能的調(diào)節(jié)作用和調(diào)控的分子機制。方法30只成年雄性Sprague Dawley(SD)大鼠(180±20 g),由南通大學(xué)實驗動物中心提供,隨機分為5組,每組6只,構(gòu)建大鼠左后肢坐骨神經(jīng)缺損1 cm模型,在損傷后0 d、1 d、4 d、7 d和14 d五個時間點處死大鼠,取近端坐骨神經(jīng)組織(0.5 cm)用于芯片分析。Trizol抽提總RNA,qRT-PCR和ISH驗證坐骨神經(jīng)損傷后五個時間點miR-9的表達變化;EdU染色、Annexin V-PE實驗、Transwell以及Wound healing assay檢測miR-9對施萬細胞增殖、凋亡和遷移的影響;不同分析預(yù)測miR-9的靶基因可能是collagen triple helix repeat-containing protein 1(CTHRC1),在luciferase報告載體上將CTHRC1的3’-UTR構(gòu)建到luciferase基因的下游,根據(jù)檢測的熒光活性進一步確定miR-9直接靶向CTHRC1 3’-UTR;qRT-PCR和Western blotting驗證近端坐骨神經(jīng)組織中CTHRC1的表達情況,并通過免疫組化判斷CTHRC1在細胞中的定位情況;使用CTHRC1 siRNA驗證CTHRC1對施萬細胞遷移的影響,并通過回復(fù)實驗確定miR-9是通過CTHRC1對施萬細胞的遷移發(fā)揮作用;使用Rac1抑制劑來確定miR-9、CTHRC1與Rac1 GTPase三者在坐骨神經(jīng)損傷后對施萬細胞遷移的調(diào)節(jié)作用;進一步在體內(nèi)驗證miR-9對施萬細胞遷移的影響。結(jié)果坐骨神經(jīng)損傷后,近端神經(jīng)中有多種miRNAs的表達發(fā)生改變;與0 d相比,miR-9在1 d、4 d、7 d和14 d呈現(xiàn)明顯的低表達;miR-9抑制施萬細胞遷移,但對細胞增殖和凋亡沒有影響;miR-9在轉(zhuǎn)錄后水平通過直接靶向CTHRC1的3’-UTR來抑制CTHRC1的表達,并且通過抑制CTHRC1的表達來抑制施萬細胞遷移;另外,miR-9抑制施萬細胞遷移是通過使CTHRC1下游的Rac1 GTPase失活;坐骨神經(jīng)損傷后在體內(nèi)注入miR-9的模擬物,近端神經(jīng)中施萬細胞遷移的數(shù)量和距離均減少了,表明miR-9在體內(nèi)也抑制了施萬細胞遷移。結(jié)論本課題通過芯片篩選和分析發(fā)現(xiàn)在坐骨神經(jīng)損傷與再生過程中表達發(fā)生改變的一些重要的miRNAs,其中miR-9呈現(xiàn)明顯的低表達,且miR-9能在體外和體內(nèi)明顯抑制施萬細胞遷移。miR-9對施萬細胞遷移的調(diào)節(jié)作用是通過在轉(zhuǎn)錄后水平抑制CTHRC1表達,并進一步使其下游的Rac1 GTPase失活來實現(xiàn)的。本課題探討了miR-9在周圍神經(jīng)損傷與再生過程中對施萬細胞遷移的調(diào)節(jié)作用,證明了miRNA可能是施萬細胞激活和表型改變的一種重要調(diào)節(jié)器,同時豐富了miRNA對周圍神經(jīng)再生的分子機制研究,并為周圍神經(jīng)的再生修復(fù)提供可參考的理論依據(jù)和新的治療靶點。
[Abstract]:Objective Regeneration after peripheral nerve injury is an extremely complex process. There are many genes and proteins in injured nerve tissue that change. MicroRNAs (miRNAs) as a kind of small non-coding RNA to regulate gene posttranscriptional level. The results provide a new model for studying the molecular mechanism of nerve injury and regeneration. Schwann cells are the main glial cells in peripheral nervous system, which can improve the microenvironment of peripheral nerve and play a very important role in the degeneration and regeneration of peripheral nerve. The aim of this study was to investigate the regulatory effect and molecular mechanism of miR-9 on Schwann cell function during sciatic nerve injury and regeneration. Methods Thirty adult male Sprague Dawley (SD) rats (180 鹵20 g),) were randomly divided into 5 groups, 6 rats in each group, and 1 cm model of sciatic nerve defect in the left hind limb of rats was established. The proximal sciatic nerve tissue (0. 5 cm) was used for microarray analysis. Total RNA,qRT-PCR and ISH extracted by Trizol were used to verify the changes of miR-9 expression at five time points after sciatic nerve injury. EdU staining, Annexin V-PE assay, Transwell and Wound healing assay were used to detect the effects of miR-9 on the proliferation, apoptosis and migration of Schwann cells. Different analyses predicted that the target gene of miR-9 might be collagen triple helix repeat-containing protein 1 (CTHRC1). The 3'-UTR of CTHRC1 was constructed into the downstream of luciferase gene in luciferase report vector. According to the fluorescence activity detected, the direct targeting of miR-9 to CTHRC1 _ (3) -UTR-UTRwas further determined. QRT-PCR and Western blotting were used to verify the expression of CTHRC1 in the proximal sciatic nerve and the localization of CTHRC1 in the cells was determined by immunohistochemistry. CTHRC1 siRNA was used to verify the effect of CTHRC1 on Schwann cell migration, and the effect of miR-9 on Schwann cell migration was confirmed by reverse-response test. The effects of miR-9,CTHRC1 and Rac1 GTPase on Schwann cell migration after sciatic nerve injury were determined by using Rac1 inhibitor, and the effect of miR-9 on Schwann cell migration was further verified in vivo. Results after sciatic nerve injury, the expression of multiple miRNAs in the proximal nerve changed, and the expression of miR-9 was significantly lower on the 1st day, 4th day, 7th day and 14th day compared with 0 day. MiR-9 inhibited the migration of Schwann cells, but had no effect on cell proliferation and apoptosis. MiR-9 inhibited the expression of CTHRC1 by directly targeting the 3'-UTR of CTHRC1 at post-transcriptional level, and inhibited the migration of Schwann cells by inhibiting the expression of CTHRC1. In addition, miR-9 inhibits Schwann cell migration by inactivating Rac1 GTPase downstream of CTHRC1. After sciatic nerve injury, the number and distance of Schwann cell migration in proximal nerve decreased after injection of miR-9 in vivo, indicating that miR-9 also inhibited Schwann cell migration in vivo. Conclusion by microarray screening and analysis, we found some important miRNAs, in the process of sciatic nerve injury and regeneration, in which the expression of miR-9 was significantly lower. MiR-9 could significantly inhibit Schwann cell migration in vitro and in vivo. The regulation of miR-9 on Schwann cell migration was achieved by inhibiting the expression of CTHRC1 at the post-transcriptional level and further inactivating its downstream Rac1 GTPase. The aim of this study was to investigate the role of miR-9 in the regulation of Schwann cell migration during peripheral nerve injury and regeneration, and to prove that miRNA may be an important regulator for Schwann cell activation and phenotypic changes. At the same time, it enriches the molecular mechanism of peripheral nerve regeneration by miRNA, and provides reference theoretical basis and new therapeutic target for the regeneration and repair of peripheral nerve.
【學(xué)位授予單位】：南通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R745
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本文編號：2313220