本文選題：急性眼高壓　切入點：RIP3　出處：《中南大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：研究受體相互作用蛋白3(Receptor Interacting Protein3, RIP3)在正常大鼠視網(wǎng)膜中的分布以及急性眼高壓早期大鼠視網(wǎng)膜RIP3表達的時空變化,初步探討急性高眼壓早期導(dǎo)致視網(wǎng)膜節(jié)細(xì)胞損傷的可能分子機制。 方法：(1)隨機挑選6只健康的成年Sprague-Dawley大鼠,3只活取視網(wǎng)膜組織勻漿后進行免疫蛋白印跡法檢驗抗體特異性,3只灌注取材之后冰凍切片進行抗體吸收實驗和免疫組織化學(xué)染色以及RIP3與Neuron-specific nuclear antigen(NeuN,神經(jīng)節(jié)細(xì)胞標(biāo)志物)、Calbindin(CB,水平細(xì)胞標(biāo)志物)、Glial fibrillary acidic protein(GFAP,星形膠質(zhì)細(xì)胞標(biāo)志物)、Glutamine synthetase(GS, Muller細(xì)胞標(biāo)志物)、Protein kinase C alpha(PKC-a,雙極細(xì)胞標(biāo)志物)、Parvalbumin(P V,無長突細(xì)胞標(biāo)志物)、Rhodopsin(視細(xì)胞標(biāo)志物)、Synaptophysin(突觸標(biāo)志物)、Integrin, alpha M(CD11b,小膠質(zhì)細(xì)胞標(biāo)志物)免疫熒光組織化學(xué)雙標(biāo)染色,分析RIP3在正常成年大鼠視網(wǎng)膜不同細(xì)胞類型中的表達分布；(2)挑選18只健康的成年Sprague-Dawley大鼠按照存活時間隨機分為6小時、12小時、24小時三個組,每組6只。一側(cè)眼球進行加壓處理、另一側(cè)眼球作為自體對照,不作任何處理。高眼壓側(cè)眼球在眼前房插針加壓,維持壓力至110毫米汞柱1小時,下架存活至相對應(yīng)的時間點后,每個小組隨機取3只大鼠注射過量的麻藥后冰上快速剝離視網(wǎng)膜組織,免疫蛋白印跡法分析RIP3的表達量。每組另外3只大鼠灌注取材用免疫熒光染色分析RIP3在視網(wǎng)膜中的表達變化。 結(jié)果：1.RIP3在正常成年大鼠視網(wǎng)膜中的免疫組織化學(xué)染色顯示：神經(jīng)纖維層和節(jié)細(xì)胞層染色最強,內(nèi)核層染色強度較弱可見顆粒狀細(xì)胞形態(tài),內(nèi)網(wǎng)層染色強度最弱,在外網(wǎng)層邊緣部分可見少量染色較淺的RIP3陽性細(xì)胞分布,外網(wǎng)層未見明顯陽性細(xì)胞染色,染色強度較弱；2.免疫熒光組織化學(xué)雙標(biāo)染色顯示：在節(jié)細(xì)胞層,RIP3的陽性產(chǎn)物與NeuN陽性產(chǎn)物存在明顯雙標(biāo)；在內(nèi)核層靠近內(nèi)網(wǎng)層邊緣部分與部分PV陽性產(chǎn)物存在明顯雙標(biāo)；在內(nèi)核層靠近外網(wǎng)層邊緣部分與部分CB陽性產(chǎn)物存在明顯雙標(biāo),此外,RIP3與PKC-a,GS陽性產(chǎn)物,GFAP陽性產(chǎn)物以及CDllb陽性產(chǎn)物呈現(xiàn)微弱的雙標(biāo)；3.在急性高眼壓處理以后,Western blot顯示RIP3表達明顯增強,表現(xiàn)為6小時、12小時的條帶明顯增粗,24小時條帶較12小時弱；免疫熒光染色顯示：急性高眼壓損傷后,損傷組與正常對照組相比RIP3陽性染色分布未見明顯變化,節(jié)細(xì)胞層的RIP3陽性染色明顯較對照組強,其中12小時染色最強,6小時和24小時的染色強度較空白對照組有所增強,但較12小時染色弱。 結(jié)論：RIP3主要分布于視網(wǎng)膜節(jié)細(xì)胞,并在部分無長突細(xì)胞和水平細(xì)胞以及膠質(zhì)細(xì)胞內(nèi)表達,而在其他類型的視網(wǎng)膜細(xì)胞未見明顯表達。急性高眼壓早期可導(dǎo)致視網(wǎng)膜節(jié)細(xì)胞RIP3的表達上調(diào)。
[Abstract]:Objective: to study the distribution of receptor interaction protein 3Receptor Interacting protein 3 (RIP3) in the retina of normal rats and the temporal and spatial changes of RIP3 expression in the retina of rats at the early stage of acute ocular hypertension. To explore the possible molecular mechanism of retinal ganglion cell injury caused by acute intraocular hypertension. Methods 1) 6 healthy adult Sprague-Dawley rats were randomly selected for immunoblotting to detect the specificity of antibodies, 3 frozen sections were used for antibody absorption test and immunological group, after the retinal homogenate was taken alive, the specific antibody was detected by Western blotting. Histochemical staining and RIP3 and Neuron-specific nuclear antigenn, ganglion cell marker Calbindinn, horizontal cell marker Glial fibrillary acidic protein RIP3, astrocytic marker Glutamine synthetaseGS, Muller cell marker protein kinase C, bipolar cell marker Parvalbumin P V. Rhodopsin (Synaptophysin, a synaptic marker, alpha M#en0# CD11b, microglia marker) was stained with double immunofluorescence histochemical staining. To analyze the expression and distribution of RIP3 in different cell types of normal adult rat retina, 18 healthy adult Sprague-Dawley rats were randomly divided into three groups according to their survival time: 6 eyes in each group were treated with compression. The other side of the eye was used as an autologous control, without any treatment. The high intraocular pressure side pressed the eyeball in the anterior chamber pin, maintained the pressure to 110 mmHg for 1 hour, and survived until the corresponding time point. Each group randomly selected three rats who were given an excessive amount of anesthetic to rapidly peel off retinal tissue on ice. The expression of RIP3 was analyzed by Western blot and the changes of RIP3 expression in retina were analyzed by immunofluorescence staining. Results 1. The immunohistochemical staining of RIP3 in the retina of normal adult rats showed that the staining intensity of nerve fiber layer and ganglion cell layer was the strongest, the staining intensity of nucleus layer was weaker than that of inner reticulum layer. A small number of RIP3 positive cells were observed in the edge of the outer reticulum layer, but there were no obvious positive cells in the outer reticulum layer. Immunofluorescence staining showed that the positive product of RIP3 and the positive product of NeuN were double labeled in the ganglion cell layer, and in the inner reticulum layer, the positive product of RIP3 was obviously double labeled with the part of PV positive product in the inner reticulum layer. In the inner layer near the edge of the outer netting layer, there was obvious double labeling between the part of CB positive product and the part of the inner layer. In addition, the GFAP positive products and CDllb positive products of RIP3 and PKC-aGS-positive products showed weak double labeling. After acute intraocular pressure treatment, the expression of RIP3 was significantly increased by Western blot. After acute intraocular pressure injury, the distribution of RIP3 positive staining in the injured group was not significantly different from that in the normal control group. The RIP3 positive staining in ganglion cell layer was significantly stronger than that in the control group. The staining intensity at 12 hours was stronger than that in the control group at 6 and 24 hours, but was weaker than that in the control group. ConclusionRIP3 is mainly distributed in retinal ganglion cells and expressed in some apogonoid cells, horizontal cells and glial cells. The expression of RIP3 in retinal ganglion cells was up-regulated at the early stage of acute intraocular pressure (IOP).
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R774.1;Q436

【共引文獻】

相關(guān)博士學(xué)位論文 前1條

1 馮珊珊;RIP3誘導(dǎo)腫瘤細(xì)胞凋亡的分子機制研究[D];中國科學(xué)技術(shù)大學(xué);2007年

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本文編號：1635702