miR-9调控施万细胞参与坐骨神经再生的机制
发布时间：2018-11-05 19:57
【摘要】：目的周围神经损伤后再生是一个极其复杂的过程,损伤的神经组织中有多种基因和蛋白的表达发生改变。microRNAs(miRNAs)作为一类小的非编码RNA在基因转录后水平发挥调控作用,其发现为研究神经损伤与再生过程中的分子机制提供了一个新的作用模式。施万细胞是周围神经系统主要的胶质细胞,能改善周围神经微环境,在周围神经损伤后的变性和再生中有着非常重要的作用。本课题旨在探讨坐骨神经损伤与再生过程中miR-9对施万细胞功能的调节作用和调控的分子机制。方法30只成年雄性Sprague Dawley(SD)大鼠(180±20 g),由南通大学实验动物中心提供,随机分为5组,每组6只,构建大鼠左后肢坐骨神经缺损1 cm模型,在损伤后0 d、1 d、4 d、7 d和14 d五个时间点处死大鼠,取近端坐骨神经组织(0.5 cm)用于芯片分析。Trizol抽提总RNA,qRT-PCR和ISH验证坐骨神经损伤后五个时间点miR-9的表达变化;EdU染色、Annexin V-PE实验、Transwell以及Wound healing assay检测miR-9对施万细胞增殖、凋亡和迁移的影响;不同分析预测miR-9的靶基因可能是collagen triple helix repeat-containing protein 1(CTHRC1),在luciferase报告载体上将CTHRC1的3’-UTR构建到luciferase基因的下游,根据检测的荧光活性进一步确定miR-9直接靶向CTHRC1 3’-UTR;qRT-PCR和Western blotting验证近端坐骨神经组织中CTHRC1的表达情况,并通过免疫组化判断CTHRC1在细胞中的定位情况;使用CTHRC1 siRNA验证CTHRC1对施万细胞迁移的影响,并通过回复实验确定miR-9是通过CTHRC1对施万细胞的迁移发挥作用;使用Rac1抑制剂来确定miR-9、CTHRC1与Rac1 GTPase三者在坐骨神经损伤后对施万细胞迁移的调节作用;进一步在体内验证miR-9对施万细胞迁移的影响。结果坐骨神经损伤后,近端神经中有多种miRNAs的表达发生改变;与0 d相比,miR-9在1 d、4 d、7 d和14 d呈现明显的低表达;miR-9抑制施万细胞迁移,但对细胞增殖和凋亡没有影响;miR-9在转录后水平通过直接靶向CTHRC1的3’-UTR来抑制CTHRC1的表达,并且通过抑制CTHRC1的表达来抑制施万细胞迁移;另外,miR-9抑制施万细胞迁移是通过使CTHRC1下游的Rac1 GTPase失活;坐骨神经损伤后在体内注入miR-9的模拟物,近端神经中施万细胞迁移的数量和距离均减少了,表明miR-9在体内也抑制了施万细胞迁移。结论本课题通过芯片筛选和分析发现在坐骨神经损伤与再生过程中表达发生改变的一些重要的miRNAs,其中miR-9呈现明显的低表达,且miR-9能在体外和体内明显抑制施万细胞迁移。miR-9对施万细胞迁移的调节作用是通过在转录后水平抑制CTHRC1表达,并进一步使其下游的Rac1 GTPase失活来实现的。本课题探讨了miR-9在周围神经损伤与再生过程中对施万细胞迁移的调节作用,证明了miRNA可能是施万细胞激活和表型改变的一种重要调节器,同时丰富了miRNA对周围神经再生的分子机制研究,并为周围神经的再生修复提供可参考的理论依据和新的治疗靶点。
[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 卤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.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R745
，
本文编号：2313220
[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 卤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.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R745
，
本文编号：2313220
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