Nogo氨基末端在视神经再生中的作用及机制研究

发布时间：2018-05-09 17:34

本文选题：Nogo氨基末端 + 整合素　；参考：《第三军医大学》2013年博士论文

【摘要】：眼睛把光投射到视网膜感受器并转化成电信号冲动，通过视神经传递到脑部而形成视觉。视网膜神经节细胞（retina ganglion cells, RGCs）的轴突形成视神经，而视神经损伤是视力丧失的原因之一。虽然视神经损伤在视力丧失中仅占一小部分比例，但其造成的视力损害是不可逆的。研究表明视神经受损后无法再生，其主要原因是视网膜神经节细胞损伤后轴突缺乏再生能力以及视网膜神经节细胞逆行退化导致节细胞凋亡。在成年哺乳动物中枢神经轴突再生失败已被证实主要是由于存在内源性抑制剂抑制轴突再生，这些抑制剂包括Nogo A、髓鞘相关性糖蛋白（Myelin associatedglycoprotein, MAG）和少突胶质细胞髓鞘糖蛋白（Oligodendrocyte myelin glycoprotein,OMGP）等。在这些内源性的抑制剂当中，Nogo A一直备受关注。Nogo A是髓磷脂来源的抑制剂，包括两个功能结构域：Nogo66和Nogo氨基末端结构域。不同的结构域可与不同的受体结合发挥不完全相同的生物学功能，Nogo66与其受体（Nogo66receptor, NgR）结合发挥双重作用，既抑制轴突再生，又在中枢神经系统神经发生过程中调节轴突生长、导向和塑形，而Nogo氨基末端只发挥抑制作用。Nogo氨基末端是否能够抑制视神经再生及其作用机制尚未见报道。整合素是一种细胞表面糖蛋白，由非共价键连接的α和β两个亚单位形成异二聚体复合物。整合素衔接细胞外基质的配体，形成粘附复合物，为肌动蛋白细胞骨架提供耦合，这对细胞的扩展、促进生长锥向远端的生长都是必须的。Nogo氨基末端通过整合素抑制细胞粘附和轴突生长。整合素αv和整合素α5广泛分布在中枢神经系统，Nogo氨基末端是否通过整合素αv和（或）整合素α5信号通路在视神经轴突生长过程中发挥作用仍需进一步研究。本课题主要进行了以下三个方面的研究：1、观察Nogo氨基末端信号通路在视觉神经系统中的表达情况。2、证实Nogo氨基末端对于视神经再生修复具有抑制作用。3、证实Nogo氨基末端通过整合素αv信号通路，而不是α5信号通路发挥抑制作用。目的:证实Nogo氨基末端对于视神经再生具有抑制效应且其通过整合素信号通路发挥作用，为治疗视神经损伤提供新的策略，，同时也为中枢神经损伤修复研究提供借鉴和参考。方法：采用免疫组织化学方法检测整合素αv、整合素α5和成簇黏附激酶（focaladhesion kinase, FAK）蛋白的表达，体外筛选针对大鼠Nogo A的小分子干扰RNA序列，重组腺相关病毒包转和纯化。原代培养视网膜神经节细胞转染rAAV2/8NC siRNA，rAAV2/8Nogo A siRNA，Nogo66竞争拮抗剂（Nep140）和Nogo氨基末端功能片段（△20）刺激，Thy1免疫荧光染色观察视网膜神经节细胞轴突长度。建立视神经钳夹伤模型，玻璃体腔注射PBS，rAAV2/8NC siRNA，rAAV2/8Nogo A siRNA，Nep140和△20，荧光金逆行标记视网膜神经节细胞存活率，GAP43染色观察再生视神经纤维情况，F VEP检测视神经功能。Western Blot检测整合素αv、整合素α5和FAK蛋白的表达。RhoA G LISA法检测RhoA活性。主要结果和结论如下： 1、免疫组织化学染色方法证实整合素αv、整合素α5以及整合素的下游关键分子FAK均可在大鼠的视皮质、视网膜以及视神经中表达。这些Nogo氨基末端信号通路关键分子表达在视觉通路上，说明Nogo氨基末端有可能通过整合素信号途径发挥作用。 2、Nogo A靶向siRNA可高效特异的下调RGCs的Nogo A的表达，而通用阴性对照序列对Nogo A的表达无影响，成功筛选出有效RNAi序列，为本研究中构建Nogo A靶向siRNA重组腺相关病毒表达载体提供了实验前提。Nogo A靶向siRNA重组腺相关病毒表达载体可有效的抑制原代培养的RGCs以及活体动物视网膜神经节细胞层的Nogo A蛋白的表达，且在动物体内Nogo A的抑制效应于病毒注射后4w即已存在，8w时仍持续，提示重组腺相关病毒已成功的整合到RGCs基因组中，从而可以发挥其长久RNA干扰作用。 3、体外实验采用RNAi完全抑制Nogo A和针对Nogo A的部分片段（Nogo66）的拮抗剂（Nep140）均可促进RGCs轴突生长，但完全抑制Nogo A后促生长作用更强，说明Nogo A上尚存在除Nogo66外的抑制片段，而给予Nogo氨基末端的功能片段△20能够明显抑制RGCs轴突生长，证实Nogo氨基末端即为Nogo A上除Nogo66外的抑制片段。 4、在体动物实验，荧光金逆行标记观察RGCs存活率，发现Nogo氨基末端对RGCs存活的抑制作用，通过生长相关蛋白43（growth associatted protein43, GAP43）免疫荧光组织化学染色观察视神经钳夹伤后视神经再生情况证实Nogo氨基末端抑制神经再生修复；F VEP检测证明Nogo氨基末端不利于视神经功能的恢复，提示Nogo氨基末端抑制视神经再生修复及功能恢复。 5、通过调控Nogo A氨基末端功能片段观察其下游信号通路蛋白整合素αv、整合素α5、FAK和RhoA的表达变化，发现Nogo氨基末端通过整合素αv信号通路发挥抑制作用。
[Abstract]:The eyes projecting light to the receptor of the retina and transforming into electrical signal impulses and passing through the optic nerve to the brain to form a vision. The axons of the retinal ganglion cell (retina ganglion cells, RGCs) form the optic nerve, and the optic nerve damage is one of the causes of loss of vision. Although the optic nerve injury is only a small part of the loss of vision. The results show that the visual impairment is irreversible. The study shows that the optic nerve is damaged and can not be regenerated, mainly due to the lack of regeneration of the axons after the retinal ganglion cell injury and the retrograde degeneration of the retinal ganglion cells leading to the apoptosis of the ganglion cells. These inhibitors include Nogo A, myelin related glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (Oligodendrocyte myelin glycoprotein, OMGP) due to the existence of endogenous inhibitors. Among these endogenous inhibitors, Nogo A has attracted much attention to.Nogo to be the pulp. The inhibitor of phospholipid source, including two functional domains: Nogo66 and Nogo amino terminal domains. Different domains can combine different receptors with different biological functions. The combination of Nogo66 and its receptor (Nogo66receptor, NgR) plays a double role, inhibiting axonal regeneration and neurogenesis in the central nervous system. During the process, the axon growth, guidance and shaping are regulated, and the mechanism of the inhibitory effect of the Nogo amino terminal only on the inhibitory effect of the.Nogo amino terminal on the optic nerve regeneration and its mechanism has not yet been reported.
Integrin is a cell surface glycoprotein, which forms an hetero two polymer complex with two subunits of non covalent bonds. Integrins connect to the extracellular matrix ligand, forming adhesion complexes and providing coupling for actin cytoskeleton, which promotes the growth of the growth cones to the distal end of the.Nogo amino group. At the end, cell adhesion and axon growth are inhibited by integrin. Integrin alpha v and integrin alpha 5 are widely distributed in the central nervous system. Whether the Nogo amino terminal through integrin alpha v and (or) integrin alpha 5 signaling pathway plays a role in the development of optic axon still needs further study.
This subject is mainly studied in three aspects: 1, to observe the expression of the Nogo amino terminal signal pathway in the visual nervous system.2, which confirms that the Nogo amino terminal has an inhibitory effect on the regenerative repair of the optic nerve, which proves that the Nogo amino terminal is inhibited by the integrin alpha v signaling pathway, but not the alpha 5 signal pathway.
Objective: to verify the inhibitory effect of Nogo amino terminal on optic nerve regeneration and its function through the integrin signaling pathway to provide a new strategy for the treatment of optic nerve injury and to provide reference and reference for the repair of central nerve injury.
Methods: the expression of integrin alpha v, integrin alpha 5 and cluster adhesion kinase (focaladhesion kinase, FAK) protein were detected by immunohistochemistry. The small molecule interference RNA sequence of rat Nogo A was screened in vitro, the recombinant adeno-associated virus was transfected and purified. The primary cultured retinal ganglion cells were transfected with rAAV2/8NC siRNA, rAAV2/8Nogo. A siRNA, Nogo66 competition antagonist (Nep140) and Nogo amino terminal functional fragment (delta 20) stimulation, Thy1 immunofluorescence staining to observe the axon length of retinal ganglion cells. Establish optic nerve clamp injury model, vitreous cavity injection PBS, rAAV2/8NC siRNA, rAAV2/8Nogo A siRNA, Delta and delta 20, fluorescent gold retrograde labeling retinal ganglion cells. The regenerated optic nerve fiber was observed by GAP43 staining, and F VEP was used to detect the optic nerve function.Western Blot to detect integrin alpha v, integrin alpha 5 and FAK protein, and.RhoA G LISA method was used to detect RhoA activity.
The main results and conclusions are as follows:
1, immunohistochemical staining showed that integrin alpha v, integrin alpha 5, and FAK, a downstream key molecule of integrin, could be expressed in the visual cortex, retina and optic nerve of the rat. These Nogo amino terminal signaling pathways are expressed in the visual pathway, indicating that the Nogo amino terminal may be used by the integrin signal pathway. Effect.
2, Nogo A targeting siRNA can efficiently and specifically downregulate the expression of Nogo A of RGCs, while the universal negative control sequence has no influence on the expression of Nogo A, and the effective RNAi sequence is successfully screened. It provides the experimental premise for the construction of Nogo A target to the recombinant adeno-related virus expression vector of siRNA recombinant adeno-related virus. The inhibition of the expression of Nogo A protein in the primary cultured RGCs and the retinal ganglion cell layer of the living animals, and the inhibition effect of Nogo A in the animal body after the virus injection is already existing and 8W still persists, suggesting that the recombinant adeno-related virus has been successfully integrated into the RGCs gene group, which can play its long-term RNA interference effect.
3, in vitro, the inhibition of Nogo A and the antagonist (Nep140) of partial fragment (Nogo66) against Nogo A (Nep140) can promote the growth of RGCs axon, but the inhibitory effect of Nogo A on the growth is stronger. It shows that there is a inhibitory fragment on Nogo A, and the functional fragment of the terminal amino terminal, delta 20, can obviously inhibit the axis of RGCs. Sudden growth confirmed that the Nogo amino terminus was the inhibitory fragment of Nogo A except Nogo66.
4, in vivo animal experiments, the survival rate of RGCs was observed by the fluorescent gold retrograde labeling, and the inhibitory effect of the Nogo amino terminal on the survival of RGCs was found. Through the growth related protein 43 (growth associatted protein43, GAP43) immunofluorescence staining to observe the regenerative condition of the optic nerve after the clamp injury of the optic nerve confirmed the regeneration of the amino terminal inhibition of Nogo. F VEP test showed that the amino terminal of Nogo was not conducive to the recovery of optic nerve function, suggesting that the amino terminal of Nogo inhibited the regeneration and functional recovery of optic nerve.
5, the expression of integrin alpha 5, FAK and RhoA was observed by regulating the functional fragment of Nogo A amino terminal protein integrin alpha, integrin alpha 5, integrin alpha, and RhoA, and the inhibitory effect of the terminal Nogo amino terminal on the integrin alpha v signaling pathway was found.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R774.6

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