SAP97在光损伤引起的大鼠视网膜水肿中的作用研究

发布时间：2018-06-12 14:33

本文选题：M(u|¨)ller细胞 + 视网膜外层水肿　；参考：《复旦大学》2010年博士论文

【摘要】：以往的研究发现膜相关鸟苷酸激酶蛋白家族在突触的可塑性以及突触蛋白的聚集方面具有重要作用。而本研究的目的就是为了明确该蛋白家族的成员之一——SAP97是否参与了Muller细胞对蓝光所造成的损伤反应。第一部分：大鼠视网膜光损伤模型的建立及视网膜外层水肿的鉴定本实验选用成年Sprague-Dawley (SD)大鼠,体重190-210g,正常对照组12只,光照后1d、2d、3d各时间点各12只。所有实验用大鼠均在12h明(20-50 Lux)以及12h暗(0-10 Lux)的循环光环境下适应7d。光照组大鼠予缝线开睑,1%阿托品扩瞳,经24h暗适应后,放入光照箱中接受24h宽谱蓝光照射,光照波长为400～440nm。箱内齐大鼠眼球水平的光照强度平均为2500Lux,箱内温度控制在22～25℃。光照结束后置于黑暗中恢复24h后送回原正常环境饲养。对照组大鼠接受48h暗适应后送回原正常环境饲养。在成功建立了大鼠视网膜光损伤模型后,我们又进一步用透射电子显微镜观察了光损伤大鼠的视网膜。结果显示：正常SD大鼠视网膜结构层次分明,视锥视杆细胞内、外节排列整齐规则,外核层排列紧密,染色均匀,细胞形态规整。而在光照后1天、2天、3天的大鼠视网膜电镜下均可观察到凋亡的细胞核。正常SD大鼠视网膜感光细胞内外节排列整齐疏松,内外节间可见有少量间隙留存,且感光细胞内节中线粒体嵴排列规则。而给予蓝光照射后,视网膜感光细胞内节中的线粒体嵴排列紊乱,出现空泡化改变；内外节肿胀,内节间间隙消失,提示存在感光细胞内水肿。另外,正常SD大鼠视网膜色素上皮连接紧密,可以观察到色素上皮细胞问的紧密连接。而光损伤后,视网膜色素上皮细胞间的紧密连接消失,色素上皮细胞排列弥散,细胞内出现融合小体,且其细胞基底膜的完整性亦受到破坏。本部分的研究发现视网膜光损伤模型中光感受器的凋亡亦伴有急性视网膜水肿,后者主要表现为感光细胞的胞内水肿及视网膜色素上皮紧密连接的破坏而引起的细胞外水肿,而这些主要分布在视网膜外层。第二部分：光损伤大鼠视网膜中Mu11er细胞的反应在明确光损伤大鼠视网膜中存在视网膜外层水肿后,我们又进一步研究了在这种病理状态下Muller细胞的反应。光照后1天、2天、3天,采用免疫荧光染色观察对照组和光照组中Muller细胞上AQP4、Kir4.1及SAP97的表达变化,并分析前二者与后者的关系；用qRT-PCR和Western-blot法对对照组和光照组大鼠视网膜中的AQP4、Kir4.1及SAP97的mRNA及蛋白表达变化进行定量分析。结果显示：随着光照后时间的延长,Kir4.1荧光染色逐渐向视网膜外层延伸。而AQP4和SAP97在正常大鼠视网膜中共分布于视网膜的外丛状层,而在光照后第3天,两者共分布于视网膜外核层；对从对照组及光损伤大鼠视网膜中新鲜分离出的Mu11er细胞进行免疫荧光染色亦发现SAP97和AQP4在Mu11er细胞的突起上表达增加；强光照射后第1天SAP97和AQP4mRNA的表达量均显著上调,而Kir4. 1mRNA的表达量在光照后第1、2天稍有下降,而在光照后第3天表达量升高。AQP4和SAP97mRNA的表达量变化较一致,而Kir4.1mRNA的表达量的升高较SAP97mRNA延迟；Western-blot显示在光照后第2、3天SAP97蛋白的表达量上升(P0.05),AQP4蛋白在光照后第3天表达量上调(P0.05),而Kir4.1的蛋白表达量在光照后第1、2、3天较对照组相比无显著统计学意义。本部分的研究发现,为了应对光损伤引起的视网膜外层水肿,Muller细胞上调了AQP4和Kir4.1在视网膜外核层的表达,这一改变与SAP97完全一致。同时AQP4和SAP97在mRNA及蛋白水平的表达变化均较一致,提示SAP97可能在Muller细胞膜上AQP4和SAP97蛋白重分布过程中发挥作用。
[Abstract]:Previous studies have found that the membrane related guanosine kinase protein family plays an important role in synaptic plasticity and the aggregation of synaptic proteins. The purpose of this study is to determine whether SAP97 is one of the members of the protein family, whether Muller cells are involved in the damage to blue light. Establishment of a membrane optical injury model and identification of retinal edema
In this experiment, adult Sprague-Dawley (SD) rats, weight 190-210g, 12 normal control group, 1D, 2D, 3D at each time point were 12. All rats were used in the cyclic light environment of 12h Ming (20-50 Lux) and 12h dark (0-10 Lux) to adapt to the suture open eyelid in the 7d. illumination group, 1% atropine dilation, and put into the light after the 24h dark adaptation. The light intensity of 24h wide spectrum blue light was received in the box. The light intensity of the eye level of the rat eyeball in the 400 ~ 440nm. box was 2500Lux, the temperature in the box was controlled at 22~25. After the light ended, the light intensity was restored to the normal environment and returned to the normal environment. The control group received the 48h dark adaptation and returned to the normal environment.
After the rat retinal light damage model was successfully established, we further observed the retina of rats with light damage by transmission electron microscope. The results showed that the retinal structure of normal SD rats was distinct, the outer segments arranged neatly, the outer nuclei were arranged closely, the staining was uniform, and the morphology of the cells was regular. The apoptotic nuclei were observed on the 1 day, 2 day and 3 day of the rat retina under electron microscope.
The inner and outer segments of the retinal photoreceptor cells in normal SD rats were arranged neatly and loosely, with a small amount of space between the internal and external nodes, and the mitochondrial crista arranged in the inner nodes of the photoreceptor cells, and the mitochondrial crista in the inner nodes of the retinal light cell was arranged in disorder and vacuolization, the internal and external nodes were swollen and the internode space disappeared after blue light irradiation. It is suggested that there is edema in the photoreceptor cells.
In addition, the retinal pigment epithelium of normal SD rats is closely connected, and the close connection of the pigment epithelial cells can be observed. After light damage, the close connection between the retinal pigment epithelial cells disappears, the pigment epithelial cells are dispersed and the fusion bodies appear in the cells, and the integrity of the cell basement membrane is also damaged.
This part of this study found that the apoptosis of photoreceptors in the retinal light damage model is accompanied by acute retinal edema, which is mainly manifested in extracellular edema caused by the intracellular edema of photoreceptor cells and the close connection of retinal pigment epithelium, which are mainly distributed in the outer layer of the retina.
The second part: light induced Mu11er cell reaction in rat retina.
We further studied the response of Muller cells in this pathological state after retinal edema in the retina of a clear light damaged rat retina. 1 days, 2 days and 3 days after light irradiation, the expression of AQP4, Kir4.1 and SAP97 on the Muller cells in the control group and the light group was observed by immunofluorescence, and the first two and the latter were analyzed. Relationship between qRT-PCR, Western-blot and mRNA and protein expression of AQP4, Kir4.1 and SAP97 in retina of control group and light group were quantitatively analyzed.
The results showed that Kir4.1 fluorescent staining gradually extended to the outer retina, while AQP4 and SAP97 were distributed in the outer plexiform layer of the retina in the normal rat retina, and third days after illumination, both of them were distributed in the outer retina of the retina, and were separated from the retina of the control group and the light injured rat retina. The expression of SAP97 and AQP4 increased on the protuberance of Mu11er cells by immunofluorescence staining in Mu11er cells, and the expression of SAP97 and AQP4mRNA increased significantly on the first day after strong light irradiation, while the expression of Kir4. 1mRNA decreased slightly at 1,2 day after light, and the expression of.AQP4 and SAP97mRNA increased at the third day after light. The expression of Kir4.1mRNA was higher than that of SAP97mRNA; Western-blot showed that the expression of SAP97 protein increased (P0.05) on day 2,3 after light, and the expression of AQP4 protein increased (P0.05) at third days after illumination, but the expression of Kir4.1 protein was not significant compared with the control group at the next day after light.
In this part, we found that in order to cope with the edema of retinal outer layer caused by light damage, Muller cells up-regulated the expression of AQP4 and Kir4.1 in the outer retina of the retina. This change is exactly the same as SAP97. Meanwhile, the expression of AQP4 and SAP97 at mRNA and protein levels are all consistent, suggesting that SAP97 may be on AQP4 and SAP97 protein on the Muller cell membrane. Play a role in the redistribution process.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R774.1

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