【作者】 霍妍；

【导师】 叶剑；

【作者基本信息】 第三军医大学 ， 眼科学， 2010， 硕士

【摘要】 背景:视神经作为中枢神经系统的一部分,其损伤后再生能力有限。眼外伤等因素引起视神经损伤,往往给伤者带来不可逆转的视功能损害,导致患者视力严重受损。因此,探讨视神经损伤再生相关问题,不仅对视功能的恢复大有裨益,也对中枢神经系统疾病的治疗有着重要借鉴意义。目前研究认为:中枢神经系统(CNS)轴突并非不能再生,外部微环境中抑制因子的作用是制约中枢神经轴突再生的关键因素之一。三种髓磷脂来源的再生抑制因子:髓磷脂相关糖蛋白(myelinassociated glycoprotein ,MAG)、少突胶质细胞髓磷脂糖蛋白(oligodendrocyte myelin glycoprotein ,OMgp)和Nogo蛋白,它们与一个共同受体NgR结合从而发挥抑制作用。中枢神经系统中的Nogo是髓磷脂中最重要的一种轴突再生抑制蛋白,Nogo–A则是Nogo最主要的形式,在中枢神经系统中由少突胶质细胞和一些神经元产生。Nogo-NgR信号通路在成年哺乳动物中枢神经系统起着抑制轴突生长、再生及突触重塑的作用。然而,Nogo-NgR信号通路在神经系统发育过程中的作用还不清楚。近期研究表明,Nogo-NgR不仅抑制轴突生长,一定条件下也参与生理塑形作用。新生大鼠视觉系统尚未发育成熟,仍有很大的可塑性,因此研究正常发育新生大鼠和视神经横断新生大鼠Nogo及其受体NgR的表达有助于研究Nogo-NgR信号通路在发育过程中所起的作用。生长相关蛋白43(GAP-43)是神经系统的特殊蛋白质,在神经生长、轴突再生、出芽、塑性过程中大量的合成,这些过程可以通过对GAP-43的检测定义。因此,国际上将GAP-43列为研究神经生长发育和损伤修复等神经可塑性的首选分子探针。目的:以正常及视神经横断新生大鼠视觉通路发育过程中GAP-43蛋白的表达为分子探针,研究Nogo-A及其受体NgR在视神经生长发育及损伤后修复过程中的作用,为进一步研究Nogo-A及其受体NgR在发育过程中作用机制提供一定的参考价值,同时也为视神经再生的研究奠定实验基础。方法:使用免疫组化和Western Blot的方法观察正常、视神经横断新生和成年大鼠视觉系统发育过程中GAP-43、Nogo-A及其受体NgR蛋白的表达情况。结果:1、免疫组化结果:GAP-43,Nogo-A及NgR在新生大鼠出生后整个发育阶段以及新生和成年大鼠视神经横断伤后的视网膜及视皮质均可检测到阳性细胞的表达。2、Western Blot结果:正常对照组SD大鼠出生后生长发育过程中GAP-43蛋白表达逐渐降低(P<0.05),新生大鼠视神经横断伤后GAP-43表达较正常发育对照组增高(P<0.05),1天变化不明显,3天开始增高,14天增高幅度达峰值,之后逐渐下降直至56天基本恢复正常水平。成年大鼠视神经横断伤后GAP-43表达较正常成年对照增高(P<0.05),1天变化不明显,3天开始增高,7天增高幅度达峰值,之后逐渐下降直至56天基本恢复正常水平,且新生大鼠视神经横断组增高的幅度显著高于成年大鼠视神经横断组。3、Western Blot结果:正常对照组SD大鼠出生后生长发育过程Nogo-A蛋白表达逐渐降低(P<0.05),新生大鼠视神经横断伤后Nogo-A表达较正常发育对照增高(P<0.05),1天变化不明显,3天开始增高,14~28天增高幅度达峰值,之后逐渐下降直至56天基本恢复正常水平。成年鼠视神经横断伤后Nogo-A表达较正常成年对照增高(P<0.05),1天变化不明显,3~7天增高幅度达峰值,14天之后逐渐下降,且新生大鼠视神经横断组增高的幅度显著高于成年大鼠视神经横断组。4、Western Blot结果:正常对照组SD大鼠出生后生长发育过程NgR蛋白表达逐渐升高(P<0.05),NgR蛋白在新生和成年SD大鼠视神经横断术后表达未见明显变化(P>0.05)。结论:1、新生大鼠视觉系统早期发育过程中GAP-43蛋白高表达,随着神经元发育成熟及突触联系的建立,GAP-43表达逐渐下降,视神经损伤修复过程中,GAP-43表达增高,提示GAP-43的表达变化与神经生长及损伤修复密切相关。2、Nogo-A在新生大鼠出生后整个发育阶段视觉系统均存在表达,并且随着大鼠的生长发育,Nogo-A表达水平呈下降趋势,提示Nogo-A与中枢神经系统生长、发育密切相关。3、以GAP-43为分子探针,新生大鼠视神经损伤后Nogo-A表达与轴突损伤修复过程GAP-43表达变化一致,即Nogo-A在损伤后修复能力较强的新生大鼠中表达较高,而在成年大鼠损伤后表达较低,Nogo-A蛋白在发育阶段损伤后的变化不同于成年时期,提示其在发育期损伤修复过程中的作用有别于成年鼠。4、对新生大鼠和成年大鼠视神经损伤后不同时间点检测,研究发现NgR在新生和成年鼠损伤后表达均无明显变化。5、随着大鼠的生长发育,NgR表达水平逐渐升高,而此时Nogo-A的表达逐渐下降,NgR在新生和成年鼠损伤后表达变化亦不同于与Nogo-A,这种配体与受体的表达不一致,推测Nogo-A可能存在除了NgR之外的受体,在机体的调节过程中发挥着不同的生物学功能。更多还原

【Abstract】 Backgroud:Optic nerve is a part of central nervous system (CNS) which axonal regeneration is limited after injury. If optic nerve is injured by ocular trauma and other factors, it will cause visual function irreversible damage and even loss vision. Therefore, researching the optic nerve regeneration is not only related for restore visual function, but also have a great significance on the treatment of CNS diseases.Now, researchers believe that CNS axon can regenerate, the inhibitors in external micro-environment are the key factors of inhibition CNS axonal regeneration. Three myelin-associated outgrowth inhibitors: Oligodendrocyte-myelin glycoprotein (OMgp), Myelin-associated glycoprotein (Mag) and Nogo bind to a common neuronal receptor Nogo receptor (NgR) to inhibit axonal growth. The most important myelin-associated outgrowth inhibitor of CNS is Nogo. Nogo-A is the most significant form of Nogo and it was produced by the oligodendrocytes and some neurons in CNS.Nogo-NgR signaling pathway has been characterized as inhibitory for axonal growth, regeneration and structural plasticity in CNS of adult mammalian. However, the function of this pathway in neural mammalian development is unclear. Recent studies demonstrated that the interaction of Nogo-NgR signaling system may also participate in the physiological plasticity. After the rat is born, its visual system is still in a period of high plasticity. Thus, knowing the expression patterns of Nogo and NgR in visual system of neonatal rats is very important for understanding their multiple functions.Growth associated protein-43(GAP-43) is a nervous tissue specific protein, and is synthesized at high levels during axonal growth in neuronal development and axonal re-growth in regeneration, axonal sprouting, an indication of axonal plasticity. These can be identified by elevated the expression of GAP-43. Therefore, GAP-43 is internationally considered to be a molecular probe of axonal sprouting and regeneration. Objective:The expression of GAP-43 was as molecular probes to study the role of Nogo-A and its receptor NgR in visual system of neonatal rats during postnatal development and trstroe after optic nerve injury. This study can offer reference value for studying the mechanism of Nogo-A and NgR during the development. Meanwhile, it provide experimental basis for studying optic nerve regeneration.Methods:Using immunohistochemistry and Western Blot method to observe the GAP-43, Nogo-A and its receptor NgR expression in development of visual system in normal, neonatal optic nerve transected and adult optic nerve transected rat.Results:1、The results of immunohistochemistry show that GAP-43, Nogo-A and NgR was found to express in retinal and visual cortex of normal control group, neonatal rats optic nerve transected group and adult rats optic nerve transected group.2、The results of Western Blot show that GAP-43 was decreasesd with the development in the normal control group, P<0.05. The expression of GAP-43 in the optic nerve transection injury group is increased compared with the normal control group, P<0.05. The expression of GAP-43 in the neonatal rats optic nerve transection group was no significant change at 1d, increased at 3d and reached peak at 14d. And in the adult rats optic nerve transection injury group it was no significant change at 1d, increased at 3d and reached peak at 7d. Then they are all gradually decreased to normal levels until 56d. GAP-43 expression in the neonatal rats optic nerve transection group is higer than the adult rats optic nerve transection group.3、The results of Western Blot show that Nogo-A was decreasesd with the development in the normal control group, P<0.05. The expression of Nogo-A in the optic nerve transection injury group is increased compared with the normal control group, P<0.05. The expression of Nogo-A in the neonatal rats optic nerve transection group was no significant change at 1d, increased at 3d and reached peak at 14~28d. And in the adult rats optic nerve transection injury group it was no significant change at 1d, increased at 3d and reached peak at 7d. Then they are all gradually decreased to normal levels until 56d. Nogo-A expression in the neonatal rats optic nerve transection group is higer than the adult rats optic nerve transection group.4、The results of Western Blot show that the expression of NgR was increasesd with the development in the normal control group, P<0.05. There is no difference on the expression of NgR between the optic nerve transection injury group and the normal control group.Conclusion:1、With neonatal rats development, the expression of GAP-43 is decreasesd in the normal group. After the optic nerve injury, GAP-43 expression was increased. These suggest that the change of GAP-43 is closely related to axonal growth and regeneration.2、Nogo-A is detected to express in neonatal rats visual system and the expression of Nogo-A is decreasesd with the development. These show that Nogo-A is closely related to the CNS growth and development.3、The expression of Nogo-A in the optic nerve injury group was in accord with the molecular probe GAP-43 expression. That means the expression of Nogo-A in the neonatal rats optic nerve injury group is higer than it in the adult rats optic nerve injury group. These suggest the role of Nogo-A in axonal repair process of neonatal rats is different from adult rats.4、There is no significant change on the expression of NgR between the optic nerve injury group and the normal control group.5、The expression of Nogo-A is decreasesd and the expression of NgR is increased with the development. And the change of NgR expressiong after injury is also different from Nogo-A expression. We speculate that Nogo-A has other recepoters which play an different role in the process of adjustment.更多还原