首页 > 硕士 > 医学 > 正文

水通道蛋白4基因敲除对视网膜光损伤的影响

Effects of AQP4 Deficiency on Light-induced Retinal Damage

作者: 专业:药理学 导师:蒋沁 年度:2009 学位:硕士  院校: 南京医科大学

Keywords

Aquaporin-4, light damage, ERG, Photoreceptor degeneration, Müller cell

        水通道蛋白(aquaporin,AQP)是一类广泛存在于真核生物细胞膜上、选择性高效转运水分子的特异孔道,对机体的水液平衡和细胞微环境的稳定发挥重要调节作用。迄今,在哺乳动物中已经发现水通道蛋白家族中的13个成员(AQP0-12)。其中,水通道蛋白4(aquaporin-4,AQP4)是主要分布在中枢神经系统(central nervous system, CNS),其次在脊髓、眼、肺、肾及胃肠道等全身组织中亦有一定量的表达。在眼内,AQP4主要表达在视网膜Müller细胞和视神经星形胶质细胞。前者占视网膜内胶质成分的90%,是唯一跨越视网膜全层的细胞,对维持视网膜内环境稳态、摄取利用神经递质和对抗自由基损伤具有重要作用,同时Müller细胞广泛参与各种急慢性视网膜疾病的发生、发展。环境和人造光源都可能是视网膜光损伤的潜在威胁。持续的强光照射容易导致视网膜光化学损伤,损伤早期视网膜功能减退,感光细胞丢失,后期内核层细胞坏死,最终导致视觉功能的丧失。此外年龄相关性黄斑变性、视网膜色素变性与视网膜光损伤也有很多相似之处。视网膜光损伤的机制和药物防治一直是眼科领域的一个重要研究课题。近年来已经取得的证据表明表达AQP4的Müller细胞在眼科生理和病理学中扮演重要的角色,生理状态下Müller细胞缺失AQP4轻微降低了视网膜电图(electroretinogram,ERG)中b-波波幅;在大鼠视网膜光损伤模型中发现AQP4蛋白表达增加。此外,在小鼠视网膜缺血模型中发现AQP4基因敲除显著延缓了视网膜内核层细胞的退变过程。由于目前尚缺乏特异性的抑制剂,研究视网膜光化学损伤中AQP4的作用需借助基因敲除小鼠这一有效的手段。而开展AQP4在视网膜光损伤中的作用的研究,还将面临对CD1遗传背景的有色小鼠视网膜光损伤光源强度条件的摸索。因此,本文工作应用AQP4基因敲除小鼠,在建立视网膜光损伤模型的基础上,考察AQP4基因敲除对视网膜在光损伤前后功能和形态的影响,进一步探讨其发生机制,为视网膜光损伤的机制研究积累学术基础,并为治疗人类眼科相关疾病提供新的策略。目的:研究AQP4基因敲除对视网膜光损伤的影响。方法:应用2月龄成年野生型(AQP4+/+)及AQP4基因缺失型(AQP4-/-)雌性小鼠,1)视网膜光损伤模型的制作:小鼠暗适应24h,光照前1%托品酰胺微弱红光下扩瞳,5min后暴露于白色荧光灯照射、光强16,000±2,000lux,每天持续12h(8:00am-20:00pm)照射,夜间休息12h,连续6天,建立视网膜光损伤模型。2)视网膜电图(ERG)检测:ERG检测AQP4+/+和AQP4-/-小鼠基础状态(control,Con)、光损伤后第一天(post-light 1 day,PL1)、第三天(post-light 3 day,PL3)和第七天(post-light 7 day,PL7)的视网膜功能。3)病理学石蜡切片H-E染色对光损伤前Con与光损伤后PL3、PL7三组视网膜内(inner nuclear layer,INL)、外核层厚度(outer nuclear layer,ONL)的形态进行定性和定量评价。4)免疫荧光化学法:观察Müller细胞特异性标志物胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)在Con、PL3各组表达强度及形态的改变。结果:1)生理状态下AQP4+/+和AQP4-/-小鼠ERG和视网膜形态均无显著差异。2)光照结束后第三天(PL3),AQP4+/+小鼠视网膜电图a、b波波幅下降,在PL7时恢复到光照前水平;AQP4-/-小鼠视网膜光损伤后视网膜a、b波在PL3消失,且到PL7时a、b波波幅未改善。AQP4+/+小鼠在光照前后视网膜形态正常,结构层次清楚,内外核层排列整齐规则,外核层细胞排列9-11层;AQP4-/-小鼠光照后PL3时视网膜结构明显破坏,表现为外核层变薄,细胞排列稀疏,细胞层数减少到2-3层,至PL7时外核层消失,内外节变短,同时内核层细胞排列紊乱,细胞丢失。3)免疫荧光化学观察GFAP染色的Müller细胞在AQP4+/+小鼠PL3时胞体变大;AQP4-/-小鼠Müller细胞胞体变大且突触向外延伸,活化较AQP4+/+小鼠显著。结论:AQP4基因缺失导致CD1小鼠视网膜外核层更易受到光导致的退行性改变;光损伤后,缺失AQP4的Müller细胞GFAP表达增加,且伸出分支向外核层(ONL)延伸,表明光损伤后反应性“胶质化”,该反应可能加速了视网膜损伤的发生。综上所述,本研究工作的创新之处在于:应用AQP4基因敲除小鼠,研究AQP4在视网膜光损伤中的作用,获得AQP4发挥神经保护的直接证据;研究结果提示AQP4可能调节Müller细胞的“胶质化”参与视网膜光损伤的保护,为光损伤的研究提供新的学术研究方向。
    Aquaporins (AQPs) are integral membrane proteins that serve as channels in the transfer of water, and in some cases, small solutes across the membrane. 1992 Agre and associates serendipitously identified a member of the water channel family (CHIP28, subsequently renamed AQP1). So far, there are 13 members of the mammalian aquaporin family (AQP0-AQP12). In the central nervous system, aquaporin-4 (AQP4) is the most abundant isoform , in addition it is localized at spinal cord、eye、lung、kidney and gastrointestinal tract. In the retina, AQP4 is expressed strongly by Müller cells and astrocytes. Müller cells are the most principal glia cell in the vertabrate’s retina. They cross the entire thickness of retina, and form a compact“neuron-glial”net with neurons. According the research on Müller cells, it was found that they exert important influence on the development, nutrition and metabolism of retinal neurons. However, Müller cells respond to a wide variety of pathological stimuli and undergo reactive gliosis. This leads to a blockade of the neuroprotective glia–neuronal interactions, which in turn causes a dramatic worsening of the survival conditions of the neurons. Further, reactive Müller cells actively contribute to neuronal cell death, by releasing toxic substances and cell death-mediating cytokines; finally, excessive glial cell proliferation causes retinal damage and detachment, which results in blindness.Light as an environmental factor has been shown to be toxic to rod photoreceptors if the retina is exposed to either high light intensities or to continuous light over a long period of time. In particular, short wavelength light is responsible for solar retinitis, playing a role in the pathogenesis of age-related macular degeneration, and (as a component of the light of ophthalmologic instruments) contributing to the development of macular edema after surgery. In response to various different pathological stimuli, Müller cells show a reactive gliosis. Recent experiments showed that excessive light may cause degenerative alterations both in the outer and inner retina. A disturbance of the Müller cell-mediated potassium and water homeostasis in the retina after exposure to blue light may contribute to the degenerative alterations in the inner retina. Meanwhile the roles of AQP4 in the ocular physical and pathological conditions have gained increased attention. Distinct roles for AQP4 in neural signal transduction and cellular swelling have been proposed. AQP4 deletion in mice was associated with mildly impaired light-induced retinal potentials. AQP4 knockout mice were protected after retinal ischemia. Motivated by this body of indirect evidence, we investigate the hypothesis that AQP4 in retinal Müller cells is involved in the pathophysiology of light-induced retinal damage by using AQP4 knockout mice.PURPOSE: To investigate the influence of AQP4 deletion on light-induced retinal damage and further the underlying mechanisms.METHODS: two-month-old female AQP4+/+ and AQP4-/- CD1 mice were divided into 4 groups: Control group, post-light 1 day group (PL1), post-light 3 days group (PL3) and post-light 7 days group (PL7). Each group contained 6 mice. All the groups except the control group were continually exposed to intense white light (16,000±2,000lux) since 8:00am to 20:00pm for 6 days. Morphologic, morphmetric, ERG and GFAP immunofluorescence studies were performed to evaluate the light-induced retinal degeneration in AQP4+/+ and AQP4-/- mice.RESULTS: 1) ERG and morphology of retinas in AQP4+/+ and AQP4-/- mice were indistinguishable before light exposure. After 6 days light exposure, the ERG of the retina in AQP4-/- mice was irreversibly impaired. The ERG was mildly reduced at 3 days after light exposure in AQP4+/+ mice, but returned to the baseline at 7 days after light exposure. The thickness of outer nuclear layers (ONL) and inner nuclear layers (INL) showed rapid degeneration in AQP4-/- mice, while there was no morphological change in the retina of AQP4+/+ mice. 2) Before light exposure, Müller cells were glial fibrillary acidic protein (GFAP) negative in both genotypic mice, only the astrocytes resided in the inner limiting membrane. At 3 days after light exposure, the soma and processes of Müller cell were GFAP-positive in AQP4-/- mice. In addition, the processes showed hypertrophy and extended from the NFL to the INL. Elevated GFAP staining in soma was also noted in AQP4+/+ mice, but the processes of Müller cell showed no significant change.CONCLUSION: AQP4 knockout resulted in hypersensitivity to the light-induced retinal degeneration, indicating AQP4 be a target for the treatment of retinal degeneration diseases and a new avenue to explore the mechanism of retinal light damage.The major contributions of the present study lie in:AQP4 knockout mice have been used to investigate the effects of AQP4 in light-induced retinal damage, which helps to obtain direct evidence that AQP4 knockout increased sensitivity to retinal light damage via modulating Müller cell gliosis.
        

水通道蛋白4基因敲除对视网膜光损伤的影响

英文缩略词表4-6
中文摘要6-9
英文摘要9-11
前言13-17
材料与方法17-20
结果20-25
讨论25-27
结语27-28
参考文献28-34
综述34-53
    参考文献47-53
附录53-54
致谢54
        下载全文需50


本文地址:

上一篇:开放Kir6.2-K_ATP通道对MPP~+所致中脑神经元损伤的保护作用及其机制研究
下一篇:Urocortin在大鼠血栓闭塞性脉管炎中的作用和对COX-2表达的影响及机制的研究

分享到: 分享水通道蛋白4基因敲除对视网膜光损伤的影响到腾讯微博           收藏
评论排行
公告