受体相互作用蛋白3在大鼠急性高眼压视网膜神经节细胞中表达变化的初步研究

发布时间：2018-03-19 19:37

本文选题：急性眼高压　切入点：RIP3　出处：《中南大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：研究受体相互作用蛋白3(Receptor Interacting Protein3, RIP3)在正常大鼠视网膜中的分布以及急性眼高压早期大鼠视网膜RIP3表达的时空变化,初步探讨急性高眼压早期导致视网膜节细胞损伤的可能分子机制。方法：(1)随机挑选6只健康的成年Sprague-Dawley大鼠,3只活取视网膜组织匀浆后进行免疫蛋白印迹法检验抗体特异性,3只灌注取材之后冰冻切片进行抗体吸收实验和免疫组织化学染色以及RIP3与Neuron-specific nuclear antigen(NeuN,神经节细胞标志物)、Calbindin(CB,水平细胞标志物)、Glial fibrillary acidic protein(GFAP,星形胶质细胞标志物)、Glutamine synthetase(GS, Muller细胞标志物)、Protein kinase C alpha(PKC-a,双极细胞标志物)、Parvalbumin(P V,无长突细胞标志物)、Rhodopsin(视细胞标志物)、Synaptophysin(突触标志物)、Integrin, alpha M(CD11b,小胶质细胞标志物)免疫荧光组织化学双标染色,分析RIP3在正常成年大鼠视网膜不同细胞类型中的表达分布；(2)挑选18只健康的成年Sprague-Dawley大鼠按照存活时间随机分为6小时、12小时、24小时三个组,每组6只。一侧眼球进行加压处理、另一侧眼球作为自体对照,不作任何处理。高眼压侧眼球在眼前房插针加压,维持压力至110毫米汞柱1小时,下架存活至相对应的时间点后,每个小组随机取3只大鼠注射过量的麻药后冰上快速剥离视网膜组织,免疫蛋白印迹法分析RIP3的表达量。每组另外3只大鼠灌注取材用免疫荧光染色分析RIP3在视网膜中的表达变化。结果：1.RIP3在正常成年大鼠视网膜中的免疫组织化学染色显示：神经纤维层和节细胞层染色最强,内核层染色强度较弱可见颗粒状细胞形态,内网层染色强度最弱,在外网层边缘部分可见少量染色较浅的RIP3阳性细胞分布,外网层未见明显阳性细胞染色,染色强度较弱；2.免疫荧光组织化学双标染色显示：在节细胞层,RIP3的阳性产物与NeuN阳性产物存在明显双标；在内核层靠近内网层边缘部分与部分PV阳性产物存在明显双标；在内核层靠近外网层边缘部分与部分CB阳性产物存在明显双标,此外,RIP3与PKC-a,GS阳性产物,GFAP阳性产物以及CDllb阳性产物呈现微弱的双标；3.在急性高眼压处理以后,Western blot显示RIP3表达明显增强,表现为6小时、12小时的条带明显增粗,24小时条带较12小时弱；免疫荧光染色显示：急性高眼压损伤后,损伤组与正常对照组相比RIP3阳性染色分布未见明显变化,节细胞层的RIP3阳性染色明显较对照组强,其中12小时染色最强,6小时和24小时的染色强度较空白对照组有所增强,但较12小时染色弱。结论：RIP3主要分布于视网膜节细胞,并在部分无长突细胞和水平细胞以及胶质细胞内表达,而在其他类型的视网膜细胞未见明显表达。急性高眼压早期可导致视网膜节细胞RIP3的表达上调。
[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).
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R774.1;Q436

【共引文献】