【作者】 张扬；

【导师】 赵家良； 陈有信； 叶俊杰； 毛进；

【作者基本信息】 中国协和医科大学 ， 眼科学， 2009， 博士

【摘要】 背景各种青光眼的共同病理学机制是视网膜神经节细胞(retina ganglion cells,RGCs)的凋亡和神经纤维的变性,从而造成不可逆的视功能损伤。由于多种因素的影响,如神经营养因子缺乏,胶质瘢痕形成以及髓鞘相关抑制蛋白再生抑制作用等,包括视神经在内的中枢神经系统(Central nervous system,CNS)损伤后难以再生。已有研究证实髓鞘相关抑制蛋白Nogo-A是阻碍CNS损伤后神经元存活和再生的重要因素之一。Nogo-A是通过与受体复合物NgR/p75/Lingo-1的结合而发挥轴突再生抑制作用的,因此研究Nogo-A及其受体NgR在正常眼和高眼压模型眼的视神经和视网膜中表达情况,观察阻滞髓鞘相关抑制蛋白的作用通路对青光眼性视神经损伤的影响,有可能为青光眼视神经损伤的治疗提出新的思路。目的1、了解髓鞘相关抑制蛋白Nogo-A及其受体NgR在正常成年大鼠视网膜和视神经中表达和分布的情况。2、观察Nogo-A及其受体NgR的mRNA和蛋白在急性和慢性高眼压模型大鼠视网膜和视神经中表达变化的情况。3、探讨NgR基因沉默对慢性高眼压模型大鼠视神经损伤后轴突再生的影响。方法1、应用免疫组织化学方法,了解Nogo-A及其受体NgR在正常成年大鼠视网膜和视神经中表达和分布的情况。2、建立急性和慢性高眼压大鼠模型。3、应用RT-PCR和Western Blot方法分别了解Nogo-A及其受体NgR在急性和慢性高眼压模型大鼠造模后不同时间视网膜和视神经中转录水平和蛋白质水平表达变化的情况。4、采用siRNA玻璃体腔内注射的方法,检测特异性沉默NgR基因表达情况,应用Western Blot方法观察轴突再生特异性蛋白GAP-43表达的变化。结果1、Nogo-A在正常成年大鼠的视神经和视网膜的神经纤维层、神经节细胞层、内丛状层、内核层、外丛状层、外核层内均有表达。NgR在正常成年大鼠的视神经和视网膜的神经纤维层、神经节细胞层、内丛状层内表达。2、急性高眼压模型大鼠的视神经和视网膜组织中Nogo-A mRNA及蛋白的表达在造模后1d、3d明显增加(与对照组相比,P＜0.05),其后7d、14d又降至正常水平。慢性高眼压模型大鼠视神经和视网膜组织中Nogo-A mRNA及蛋白的表达在造模后3d即有增加,7d后增加显著,并持续至造模后14d和28d(与对照组相比,P＜0.05)。急性和慢性高眼压模型大鼠造模后不同时间,NgR mRNA和蛋白在视神经和视网膜组织中的表达未见明显变化。3、在慢性高眼压模型大鼠中,于玻璃体腔内注射特异siRNA干扰NgR基因表达,在mRNA及蛋白翻译水平都实现了基因沉默,轴突再生特异性蛋白GAP-43的表达上调。结论1、Nogo-A及其受体NgR在正常成年大鼠的视神经和视网膜中广泛分布。2、急性和慢性高眼压可以促使大鼠Nogo-A mRNA和蛋白的表达上调,表明Nogo-A在高眼压导致的视神经损伤的神经再生过程中发挥着重要作用。3、在急性和慢性高眼压模型大鼠中,NgR mRNA和蛋白的表达未见明显改变。但是化学合成的NgR特异性siRNA能够抑制慢性高眼压模型大鼠视网膜中NgRmRNA和蛋白的表达水平,使视网膜轴突再生特异性蛋白GAP-43的表达上调,促进高眼压性视神经损伤的神经再生。表明NgR在高眼压性视神经损伤中发挥作用。NgR siRNA有可能成为未来青光眼视神经保护药物。更多还原

【Abstract】 BackgroundThe pathological mechanism of glaucomatous optic neuropathy is progressive death of retina ganglion cells and optic nerve fiber degeneration,which leads to irreversible damage.The regeneration of damaged central nervous system,imcluding optic nerve, was difficulty achieved since series of factors,such as inhibitors of axonal regeneration which are present in myelin,a lack of neurotrophic factors and formation of the glia scar. Some studies have showed that the regeneration of central nervous system was influenced by myelin associated protein Nogo-A,which functioned by connecting with its receptor-complex NgR/p75/Lingo-1.Thus,finding the expression of Nogo-A and NgR in the retina and optical nerve of normal and glaucomatous models would make a new way of the rehabilitation of glaucomatous optic nerve.Objective1.To investigate the expression and distribution of Nogo-A and its receptor NgR in the retina and optic nerve of normal rats.2.To evaluate the expressive variation of Nogo-A and NgR in the retina and optic nerve in the retina and optic nerve in rat model with acute and chronic ocular hypertension.3.To explore the effect in promoting axon regeneration of optic nerve by intravitreal injection siRNA knocking down the NgR protein expression in rat with chronic ocular hypertension.Materials and Methods1.Immunohistochemistry technique was used to assay the expression and distribution of Nogo-A and its receptor NgR in the retina and optic nerve.2.Establish rat model with acute and chronic ocular hyperrention.3.Reverse-transcription-polymerase chain reaction(RT-PCR)and Western Blot methods were used to evaluate the expressive varieties of Nogo-A and NgR in the retina and optic nerve in the retina and optic nerve in rat model with acute and chronic ocular hypertension.4.The NgR mRNA and protein were evaluated by PT-PCR and Western Blot after the intravitreal injection of NgR siRNA,Western Blot was also used to evaluate the expression of GAP-43 protein. Results1.The expression of Nogo-A was found in the optic nerve,retinal ganglion cells layer, nerve fiber layer,inner nuclear layer,inner plexiform layer,outer plexiform layer and outer nuclear layer.The expression of NgR was found in the optic nerve,retinal ganglion cells layer,nerve fiber layer and inner plexiform layer.2.Rat models with acute and chronic ocular hypertension were successfully established.3.In the retina and optic nerve in rat model with acute ocular hypertension,the significant upregulation of the level of Nogo-A mRNA and protein was found at 1d,3d after the model establishment(P＜0.05 in comparing with control group),and then decrease to the nomal level at 7d and 14d after the model establishment.In the retina and optic nerve in rat model with chronic ocular hypertension,the level of Nogo-A mRNA and protein were found to increase at 3d and 7d after the model establishment,and the increase remained significantly at 14d and 28d after the model establishment(P＜0.05 in comparing with control group).The level of NgR in the ratina and optic nerve in rat with the acute and chronic ocular hypertension was not found to change significantly.4.In the retina in rat model with chronic ocular hypertension,the expression of NgR mRNA was knocked down and NgR protein was suppressed after the intravitreal injection of NgR siRNA demonstrated by using RT-PCR and Western Blot.The expression of GAP-43 protein increased after the intravitreal injection of NgR siRNA compared with control.Conclusions1.Nogo-A and NgR were widely distributed in the retina and optic nerve of normal rats.2.The change of expression of Nogo-A mRNA and protein in the retina was associated with the elevated ocular pressure.The dramatically increased Nogo-A indicated that Nogo-A may play a primary role in obstructing regeneration of optic nerve.3.The expression of NgR in the retina and optic nerve in rat model with acute and chronic ocular hypertension was not significantly changed.In the retina in rat model with chronic ocular hypertension,the level of NgR mRNA expression was knocked down by the siRNA,and NgR protein was suppressed too.NgR may also play an important effect in the mechanism of inhibiting the axon regeneration after the ocular hypertension.NgR siRNA could improve the expression GAP-43 which may promote the axon regeneration of injured optic nerve.NgR siRNA would be an effective method in the treatment of glaucomatous optic neuropathy.更多还原