本文選題：Nogo氨基末端 + 整合素��； 參考：《第三軍醫(yī)大學》2013年博士論文

【摘要】：眼睛把光投射到視網(wǎng)膜感受器并轉(zhuǎn)化成電信號沖動，通過視神經(jīng)傳遞到腦部而形成視覺。視網(wǎng)膜神經(jīng)節(jié)細胞（retina ganglion cells, RGCs）的軸突形成視神經(jīng)，而視神經(jīng)損傷是視力喪失的原因之一。雖然視神經(jīng)損傷在視力喪失中僅占一小部分比例，但其造成的視力損害是不可逆的。研究表明視神經(jīng)受損后無法再生，其主要原因是視網(wǎng)膜神經(jīng)節(jié)細胞損傷后軸突缺乏再生能力以及視網(wǎng)膜神經(jīng)節(jié)細胞逆行退化導致節(jié)細胞凋亡。在成年哺乳動物中樞神經(jīng)軸突再生失敗已被證實主要是由于存在內(nèi)源性抑制劑抑制軸突再生，這些抑制劑包括Nogo A、髓鞘相關(guān)性糖蛋白（Myelin associatedglycoprotein, MAG）和少突膠質(zhì)細胞髓鞘糖蛋白（Oligodendrocyte myelin glycoprotein,OMGP）等。在這些內(nèi)源性的抑制劑當中，Nogo A一直備受關(guān)注。Nogo A是髓磷脂來源的抑制劑，包括兩個功能結(jié)構(gòu)域：Nogo66和Nogo氨基末端結(jié)構(gòu)域。不同的結(jié)構(gòu)域可與不同的受體結(jié)合發(fā)揮不完全相同的生物學功能，Nogo66與其受體（Nogo66receptor, NgR）結(jié)合發(fā)揮雙重作用，既抑制軸突再生，又在中樞神經(jīng)系統(tǒng)神經(jīng)發(fā)生過程中調(diào)節(jié)軸突生長、導向和塑形，而Nogo氨基末端只發(fā)揮抑制作用。Nogo氨基末端是否能夠抑制視神經(jīng)再生及其作用機制尚未見報道。 整合素是一種細胞表面糖蛋白，由非共價鍵連接的α和β兩個亞單位形成異二聚體復合物。整合素銜接細胞外基質(zhì)的配體，形成粘附復合物，為肌動蛋白細胞骨架提供耦合，這對細胞的擴展、促進生長錐向遠端的生長都是必須的。Nogo氨基末端通過整合素抑制細胞粘附和軸突生長。整合素αv和整合素α5廣泛分布在中樞神經(jīng)系統(tǒng)，Nogo氨基末端是否通過整合素αv和（或）整合素α5信號通路在視神經(jīng)軸突生長過程中發(fā)揮作用仍需進一步研究。 本課題主要進行了以下三個方面的研究：1、觀察Nogo氨基末端信號通路在視覺神經(jīng)系統(tǒng)中的表達情況。2、證實Nogo氨基末端對于視神經(jīng)再生修復具有抑制作用。3、證實Nogo氨基末端通過整合素αv信號通路，而不是α5信號通路發(fā)揮抑制作用。 目的:證實Nogo氨基末端對于視神經(jīng)再生具有抑制效應(yīng)且其通過整合素信號通路發(fā)揮作用，為治療視神經(jīng)損傷提供新的策略，，同時也為中樞神經(jīng)損傷修復研究提供借鑒和參考。 方法：采用免疫組織化學方法檢測整合素αv、整合素α5和成簇黏附激酶（focaladhesion kinase, FAK）蛋白的表達，體外篩選針對大鼠Nogo A的小分子干擾RNA序列，重組腺相關(guān)病毒包轉(zhuǎn)和純化。原代培養(yǎng)視網(wǎng)膜神經(jīng)節(jié)細胞轉(zhuǎn)染rAAV2/8NC siRNA，rAAV2/8Nogo A siRNA，Nogo66競爭拮抗劑（Nep140）和Nogo氨基末端功能片段（△20）刺激，Thy1免疫熒光染色觀察視網(wǎng)膜神經(jīng)節(jié)細胞軸突長度。建立視神經(jīng)鉗夾傷模型，玻璃體腔注射PBS，rAAV2/8NC siRNA，rAAV2/8Nogo A siRNA，Nep140和△20，熒光金逆行標記視網(wǎng)膜神經(jīng)節(jié)細胞存活率，GAP43染色觀察再生視神經(jīng)纖維情況，F(xiàn) VEP檢測視神經(jīng)功能。Western Blot檢測整合素αv、整合素α5和FAK蛋白的表達。RhoA G LISA法檢測RhoA活性。 主要結(jié)果和結(jié)論如下： 1、免疫組織化學染色方法證實整合素αv、整合素α5以及整合素的下游關(guān)鍵分子FAK均可在大鼠的視皮質(zhì)、視網(wǎng)膜以及視神經(jīng)中表達。這些Nogo氨基末端信號通路關(guān)鍵分子表達在視覺通路上，說明Nogo氨基末端有可能通過整合素信號途徑發(fā)揮作用。 2、Nogo A靶向siRNA可高效特異的下調(diào)RGCs的Nogo A的表達，而通用陰性對照序列對Nogo A的表達無影響，成功篩選出有效RNAi序列，為本研究中構(gòu)建Nogo A靶向siRNA重組腺相關(guān)病毒表達載體提供了實驗前提。Nogo A靶向siRNA重組腺相關(guān)病毒表達載體可有效的抑制原代培養(yǎng)的RGCs以及活體動物視網(wǎng)膜神經(jīng)節(jié)細胞層的Nogo A蛋白的表達，且在動物體內(nèi)Nogo A的抑制效應(yīng)于病毒注射后4w即已存在，8w時仍持續(xù)，提示重組腺相關(guān)病毒已成功的整合到RGCs基因組中，從而可以發(fā)揮其長久RNA干擾作用。 3、體外實驗采用RNAi完全抑制Nogo A和針對Nogo A的部分片段（Nogo66）的拮抗劑（Nep140）均可促進RGCs軸突生長，但完全抑制Nogo A后促生長作用更強，說明Nogo A上尚存在除Nogo66外的抑制片段，而給予Nogo氨基末端的功能片段△20能夠明顯抑制RGCs軸突生長，證實Nogo氨基末端即為Nogo A上除Nogo66外的抑制片段。 4、在體動物實驗，熒光金逆行標記觀察RGCs存活率，發(fā)現(xiàn)Nogo氨基末端對RGCs存活的抑制作用，通過生長相關(guān)蛋白43（growth associatted protein43, GAP43）免疫熒光組織化學染色觀察視神經(jīng)鉗夾傷后視神經(jīng)再生情況證實Nogo氨基末端抑制神經(jīng)再生修復；F VEP檢測證明Nogo氨基末端不利于視神經(jīng)功能的恢復，提示Nogo氨基末端抑制視神經(jīng)再生修復及功能恢復。 5、通過調(diào)控Nogo A氨基末端功能片段觀察其下游信號通路蛋白整合素αv、整合素α5、FAK和RhoA的表達變化，發(fā)現(xiàn)Nogo氨基末端通過整合素αv信號通路發(fā)揮抑制作用。
[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.

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R774.6

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