本文選題：M(u|¨)ller細胞 + 視網膜外層水腫�。� 參考：《復旦大學》2010年博士論文

【摘要】：以往的研究發(fā)現膜相關鳥苷酸激酶蛋白家族在突觸的可塑性以及突觸蛋白的聚集方面具有重要作用。而本研究的目的就是為了明確該蛋白家族的成員之一——SAP97是否參與了Muller細胞對藍光所造成的損傷反應。第一部分：大鼠視網膜光損傷模型的建立及視網膜外層水腫的鑒定 本實驗選用成年Sprague-Dawley (SD)大鼠,體重190-210g,正常對照組12只,光照后1d、2d、3d各時間點各12只。所有實驗用大鼠均在12h明(20-50 Lux)以及12h暗(0-10 Lux)的循環(huán)光環(huán)境下適應7d。光照組大鼠予縫線開瞼,1%阿托品擴瞳,經24h暗適應后,放入光照箱中接受24h寬譜藍光照射,光照波長為400～440nm。箱內齊大鼠眼球水平的光照強度平均為2500Lux,箱內溫度控制在22～25℃。光照結束后置于黑暗中恢復24h后送回原正常環(huán)境飼養(yǎng)。對照組大鼠接受48h暗適應后送回原正常環(huán)境飼養(yǎng)。 在成功建立了大鼠視網膜光損傷模型后,我們又進一步用透射電子顯微鏡觀察了光損傷大鼠的視網膜。結果顯示：正常SD大鼠視網膜結構層次分明,視錐視桿細胞內、外節(jié)排列整齊規(guī)則,外核層排列緊密,染色均勻,細胞形態(tài)規(guī)整。而在光照后1天、2天、3天的大鼠視網膜電鏡下均可觀察到凋亡的細胞核。 正常SD大鼠視網膜感光細胞內外節(jié)排列整齊疏松,內外節(jié)間可見有少量間隙留存,且感光細胞內節(jié)中線粒體嵴排列規(guī)則。而給予藍光照射后,視網膜感光細胞內節(jié)中的線粒體嵴排列紊亂,出現空泡化改變；內外節(jié)腫脹,內節(jié)間間隙消失,提示存在感光細胞內水腫。 另外,正常SD大鼠視網膜色素上皮連接緊密,可以觀察到色素上皮細胞問的緊密連接。而光損傷后,視網膜色素上皮細胞間的緊密連接消失,色素上皮細胞排列彌散,細胞內出現融合小體,且其細胞基底膜的完整性亦受到破壞。 本部分的研究發(fā)現視網膜光損傷模型中光感受器的凋亡亦伴有急性視網膜水腫,后者主要表現為感光細胞的胞內水腫及視網膜色素上皮緊密連接的破壞而引起的細胞外水腫,而這些主要分布在視網膜外層。 第二部分：光損傷大鼠視網膜中Mu11er細胞的反應 在明確光損傷大鼠視網膜中存在視網膜外層水腫后,我們又進一步研究了在這種病理狀態(tài)下Muller細胞的反應。光照后1天、2天、3天,采用免疫熒光染色觀察對照組和光照組中Muller細胞上AQP4、Kir4.1及SAP97的表達變化,并分析前二者與后者的關系；用qRT-PCR和Western-blot法對對照組和光照組大鼠視網膜中的AQP4、Kir4.1及SAP97的mRNA及蛋白表達變化進行定量分析。 結果顯示：隨著光照后時間的延長,Kir4.1熒光染色逐漸向視網膜外層延伸。而AQP4和SAP97在正常大鼠視網膜中共分布于視網膜的外叢狀層,而在光照后第3天,兩者共分布于視網膜外核層；對從對照組及光損傷大鼠視網膜中新鮮分離出的Mu11er細胞進行免疫熒光染色亦發(fā)現SAP97和AQP4在Mu11er細胞的突起上表達增加；強光照射后第1天SAP97和AQP4mRNA的表達量均顯著上調,而Kir4. 1mRNA的表達量在光照后第1、2天稍有下降,而在光照后第3天表達量升高。AQP4和SAP97mRNA的表達量變化較一致,而Kir4.1mRNA的表達量的升高較SAP97mRNA延遲；Western-blot顯示在光照后第2、3天SAP97蛋白的表達量上升(P0.05),AQP4蛋白在光照后第3天表達量上調(P0.05),而Kir4.1的蛋白表達量在光照后第1、2、3天較對照組相比無顯著統(tǒng)計學意義。 本部分的研究發(fā)現,為了應對光損傷引起的視網膜外層水腫,Muller細胞上調了AQP4和Kir4.1在視網膜外核層的表達,這一改變與SAP97完全一致。同時AQP4和SAP97在mRNA及蛋白水平的表達變化均較一致,提示SAP97可能在Muller細胞膜上AQP4和SAP97蛋白重分布過程中發(fā)揮作用。
[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.
【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R774.1

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