本文選題：M(u|¨)ller細(xì)胞 + 視網(wǎng)膜外層水腫��； 參考：《復(fù)旦大學(xué)》2010年博士論文

【摘要】：以往的研究發(fā)現(xiàn)膜相關(guān)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(xì)胞對(duì)藍(lán)光所造成的損傷反應(yīng)。第一部分：大鼠視網(wǎng)膜光損傷模型的建立及視網(wǎng)膜外層水腫的鑒定 本實(shí)驗(yàn)選用成年Sprague-Dawley (SD)大鼠,體重190-210g,正常對(duì)照組12只,光照后1d、2d、3d各時(shí)間點(diǎn)各12只。所有實(shí)驗(yàn)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(yīng)7d。光照組大鼠予縫線開瞼,1%阿托品擴(kuò)瞳,經(jīng)24h暗適應(yīng)后,放入光照箱中接受24h寬譜藍(lán)光照射,光照波長(zhǎng)為400～440nm。箱內(nèi)齊大鼠眼球水平的光照強(qiáng)度平均為2500Lux,箱內(nèi)溫度控制在22～25℃。光照結(jié)束后置于黑暗中恢復(fù)24h后送回原正常環(huán)境飼養(yǎng)。對(duì)照組大鼠接受48h暗適應(yīng)后送回原正常環(huán)境飼養(yǎng)。 在成功建立了大鼠視網(wǎng)膜光損傷模型后,我們又進(jìn)一步用透射電子顯微鏡觀察了光損傷大鼠的視網(wǎng)膜。結(jié)果顯示：正常SD大鼠視網(wǎng)膜結(jié)構(gòu)層次分明,視錐視桿細(xì)胞內(nèi)、外節(jié)排列整齊規(guī)則,外核層排列緊密,染色均勻,細(xì)胞形態(tài)規(guī)整。而在光照后1天、2天、3天的大鼠視網(wǎng)膜電鏡下均可觀察到凋亡的細(xì)胞核。 正常SD大鼠視網(wǎng)膜感光細(xì)胞內(nèi)外節(jié)排列整齊疏松,內(nèi)外節(jié)間可見有少量間隙留存,且感光細(xì)胞內(nèi)節(jié)中線粒體嵴排列規(guī)則。而給予藍(lán)光照射后,視網(wǎng)膜感光細(xì)胞內(nèi)節(jié)中的線粒體嵴排列紊亂,出現(xiàn)空泡化改變；內(nèi)外節(jié)腫脹,內(nèi)節(jié)間間隙消失,提示存在感光細(xì)胞內(nèi)水腫。 另外,正常SD大鼠視網(wǎng)膜色素上皮連接緊密,可以觀察到色素上皮細(xì)胞問的緊密連接。而光損傷后,視網(wǎng)膜色素上皮細(xì)胞間的緊密連接消失,色素上皮細(xì)胞排列彌散,細(xì)胞內(nèi)出現(xiàn)融合小體,且其細(xì)胞基底膜的完整性亦受到破壞。 本部分的研究發(fā)現(xiàn)視網(wǎng)膜光損傷模型中光感受器的凋亡亦伴有急性視網(wǎng)膜水腫,后者主要表現(xiàn)為感光細(xì)胞的胞內(nèi)水腫及視網(wǎng)膜色素上皮緊密連接的破壞而引起的細(xì)胞外水腫,而這些主要分布在視網(wǎng)膜外層。 第二部分：光損傷大鼠視網(wǎng)膜中Mu11er細(xì)胞的反應(yīng) 在明確光損傷大鼠視網(wǎng)膜中存在視網(wǎng)膜外層水腫后,我們又進(jìn)一步研究了在這種病理狀態(tài)下Muller細(xì)胞的反應(yīng)。光照后1天、2天、3天,采用免疫熒光染色觀察對(duì)照組和光照組中Muller細(xì)胞上AQP4、Kir4.1及SAP97的表達(dá)變化,并分析前二者與后者的關(guān)系；用qRT-PCR和Western-blot法對(duì)對(duì)照組和光照組大鼠視網(wǎng)膜中的AQP4、Kir4.1及SAP97的mRNA及蛋白表達(dá)變化進(jìn)行定量分析。 結(jié)果顯示：隨著光照后時(shí)間的延長(zhǎng),Kir4.1熒光染色逐漸向視網(wǎng)膜外層延伸。而AQP4和SAP97在正常大鼠視網(wǎng)膜中共分布于視網(wǎng)膜的外叢狀層,而在光照后第3天,兩者共分布于視網(wǎng)膜外核層；對(duì)從對(duì)照組及光損傷大鼠視網(wǎng)膜中新鮮分離出的Mu11er細(xì)胞進(jìn)行免疫熒光染色亦發(fā)現(xiàn)SAP97和AQP4在Mu11er細(xì)胞的突起上表達(dá)增加；強(qiáng)光照射后第1天SAP97和AQP4mRNA的表達(dá)量均顯著上調(diào),而Kir4. 1mRNA的表達(dá)量在光照后第1、2天稍有下降,而在光照后第3天表達(dá)量升高。AQP4和SAP97mRNA的表達(dá)量變化較一致,而Kir4.1mRNA的表達(dá)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(dá)量上升(P0.05),AQP4蛋白在光照后第3天表達(dá)量上調(diào)(P0.05),而Kir4.1的蛋白表達(dá)量在光照后第1、2、3天較對(duì)照組相比無顯著統(tǒng)計(jì)學(xué)意義。 本部分的研究發(fā)現(xiàn),為了應(yīng)對(duì)光損傷引起的視網(wǎng)膜外層水腫,Muller細(xì)胞上調(diào)了AQP4和Kir4.1在視網(wǎng)膜外核層的表達(dá),這一改變與SAP97完全一致。同時(shí)AQP4和SAP97在mRNA及蛋白水平的表達(dá)變化均較一致,提示SAP97可能在Muller細(xì)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(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R774.1

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