【作者】 严肖啸；

【导师】 赵平；

【作者基本信息】 河北医科大学 ， 眼科学， 2010， 硕士

【摘要】 目的:青光眼是临床最常见致盲眼病之一,发病机制尚不清楚,其中青光眼免疫学发病机制逐渐引起人们的关注。视网膜小胶质细胞作为中枢神经系统最具代表性的免疫细胞已成为研究热点,其视神经保护与毒性的双重作用归因于它对一系列病变做出反应后所能合成和分泌的物质不同,因此针对视网膜小胶质细胞双重作用的药物研究成为治疗青光眼的切入点。有研究表明雌激素具有免疫细胞凋亡的调节作用,亦有视神经保护作用,故本实验通过建立大鼠急性高眼压模型,观察雌激素对视网膜小胶质细胞的活化、诱导型一氧化碳合酶(iNOS)的表达影响及对视神经节细胞的保护,为今后治疗青光眼提供实验依据。方法:SD大鼠35只雄性(全部采用雄性大鼠的目的是尽量减少内源性雌激素对实验的影响),无眼疾,体重200±20 g,分3组,Ⅰ组即正常组5只(10眼),余30只均采用前房加压灌注法将左眼制成急性高眼压模型(以下统称模型眼),右眼为自身对照眼(以下统称为自身对照眼),再将这30只分为Ⅱ组即雌激素腹腔注射组15只和Ⅲ组即消毒橄榄油注射组15只。最终弃去实验中突然卒死及不符合标准的眼球,每组各保留10只眼球固定后行石蜡切片,应用免疫组化技术分析视网膜小胶质细胞的表达(细胞表面标记物为CD11b)及其iNOS的表达,以及视神经节细胞的凋亡。计算机图像分析软件对图像中的阳性反应部位进行光密度分析,三者半定量测定结果均以均数±标准差(Mean±SD)表示,应用SPSS统计分析软件进行统计学分析结果:1.组织病理学变化:1.1CD11b阳性小胶质细胞:Ⅰ组可见小胶质细胞淡染,呈棕黄色、分枝状,胞体小,具有伸向各个方向的细小突起。Ⅱ、Ⅲ组模型眼活化的小胶质细胞数量增加,突起变短、变粗,胞体增大、变圆,并聚集于凋亡的节细胞及细胞碎片周围。1.2视神经节细胞:Ⅰ组视网膜神经节细胞呈单层排列,无阳性细胞。Ⅱ、Ⅲ组的自身对照眼与I组相同。Ⅱ、Ⅲ组的模型眼光镜下可见视网膜神经节细胞数目明显减少,视网膜神经节细胞层呈空泡样改变,部分细胞出现核溶解、核染色浅淡、胞浆染色浅淡。Ⅱ组模型眼视网膜神经节细胞数目减少,形态大致正常,神经节细胞层结构基本正常。2.小胶质细胞免疫组织化学分析(即小胶质细胞的活化及iNOS的表达):3组CD11b阳性细胞数均可检测到,Ⅰ组未见到iNOS阳性细胞,Ⅱ、Ⅲ组模型眼CD11b阳性细胞及iNOS阳性细胞增多,均高于Ⅰ组及自身对照眼(P<0.05)。Ⅱ组模型眼染色CD11b阳性细胞及iNOS阳性细胞数少于Ⅲ组模型眼阳性细胞数(P<0.05)。3.视神经节细胞TUNEL原位凋亡监测:染色阳性的视网膜节细胞(RGCs)在3组动物眼球上均可检测到,Ⅰ组及Ⅱ、Ⅲ组的自身对照眼TUNEL染色阳性的RGCs数最少。Ⅱ、Ⅲ组模型眼上TUNEL染色阳性的RGCs稍多,阳性细胞数均高于Ⅰ组及自身对照眼(P<0.05)。Ⅱ组模型眼TUNEL染色阳性细胞数少于Ⅲ组模型眼阳性细胞数(P<0.05)。同时证实小胶质细胞的活化及其iNOS的表达均与节细胞凋亡指数呈正相关。结论:1.通过分析Ⅱ、Ⅲ组模型眼与Ⅰ组及自身对照眼CD11b及iNOS的表达(P<0.05),证实高眼压下小胶质细胞活化,并表达iNOS。2.Ⅱ、Ⅲ组模型眼的CD11b及iNOS表达差异有统计学意义(P<0.05),表明雌激素能够抑制小胶质细胞的活化,减少iNOS的表达,从而减少合成NO神经毒性物质。3.NO具有神经毒性,。4.RGCs的TUNEL检测表明雌激素能够减少节细胞的凋亡,从而保护视神经节细胞。5.3组CD11b的表达、iNOS表达均与TUNEL的表达呈直线正相关,说明在高眼压下小胶质细胞对节细胞的凋亡起到了调节作用。更多还原

【Abstract】 Objective: Glaucoma is one of the most frequent growing blind oculopathies, and its pathogenesis is not clear. Now immunological pathogenesis of glaucoma is getting attention gradually.Retinal microglial cells has become the hot reach pot as the most representative immunocyte of the central nervous system . As the different secretion of microglias, they have nerve protection and neurotoxicity. Therefore, the drug research to RMC becomes an entry point for the treatment of glaucoma . Studies have shown thatβ-estradiol has a role in the regulation of apoptosis in immunocytes and neuroprotection.This experiment was that observe effects ofβ-estradiol on activation of retinal microglia, expression of inducible nitric oxide synthase(iNOS) and effects on the protection of retinal ganglion cells (RGC)through the establishment of rat acute ocular hypertention model in order to provide experimental basis for future treatment of glaucoma.Methods: 35 SD male rats without eye diseases , each weighing 200 to 220g, were divided into 3 groups: groupⅠ:normal group(5rats,10eyes),groupⅡ:β-estradiol solution treated group (15rats),groupⅢ:balanced disinfected olive solution treated group(15 rats).Rats in groupⅡandⅢwere induced in left eye byforcing perfusion of balanced disinfected olive solution into the anterior chamber as acute ocular hypertention model.Right eyes were self-control eyes.Not standard eyeballswere discard and 10 eyeballs were selected and fixed in each group.The RMS were detected by immunohistological technique to observe the expression of RMC and iNOS and the effects on the protection of RGCs.The apoptosis cells were detected by TdT-dUDP terminal nick end-labeling (TUNEL) technique.The computer image analysis system was used to analysis the expression of RMC , iNOS and the apoptosis cells of RGCs in retina after immunohistochemistry stain.SPSS 13.0 was used to analyse data of this study.Results: 1.The change of histopathology1.1 CD11b-positive microglias:in groupⅠand the self–control eyes of groupⅡand groupⅢ, the pale staining cells were brown and banching. The cell bodies were small and they had dendritic extensions. In the model eyes of groupⅡand groupⅢ,the cells number is more. Ramified microglias changed their morphology to reactive or amoeboid forms accumulating in the apoptosis RGCs and cells segments.1.2 RGCs: in groupⅠand the self–control eyes of groupⅡand groupⅢ,RGCs were in singl arrangement,and there was no positive cell. In the model eyes of groupⅡand groupⅢ, the number of RGCs reduced evidently,and the ganglion cells layer(GCL) became vacuolated change. In the model eyes of groupⅡ, the number of RGCs reduced,but the structure of GCL was normal.2. The immunohistochemistry analysis of retinal microglial cells(the activation of retinal microglial cells and the expression iNOS): CD11b, the surface marker of microglia, expressed in all the group. InⅠgroup ,iNOS-positive cells didn’t express. .The CD11b -positive cells and iNOS-positive cells of model eyes in groupⅡandⅢwere more than that of group I and self-control eyes,there were significant diferences (P<0.05) .But the CD11b-positive cells and iNOS-positive cells of model eyes in groupⅡwere less than that of groupⅢ,there were significant diferences (P<0.05). In all,there was statistical difference between model and self-control eyes both in the 2 groups and the significant difference of the model eyes between the two groups was also found (P<0.05).3. The TUNEL test of RGCs: The TUNEL-positive cells expressed in all the group. The TUNEL-positive cells of self-control eyes in groupⅡandⅢare lest,so was groupⅠ.The TUNEL-positive cells of model eyes in groupⅡandⅢwere more than that of groupⅠand self-control eyes,there were significant diferences (P<0.05).But the TUNEL-positive cells of model eyes in groupⅡwere less than that of groupⅢ,there were significant diferences (P<0.05).Meanwhile confirmed that the expression of CD11b-positive microglias was assumed linear correlation,so did the expression of iNOS and the apoptosis of RGCs.Conclusion:1. By analyzing the expression of CD11b and iNOS of the model eyes of groupⅡ,Ⅲ, groupⅠand self-control eyes (P <0.05), the retinal microglial cells activate and express iNOS in acute ocular hypertention.2. The expression of CD11b and iNOS of the model eyes of groupⅡ,Ⅲwas significant,soβ-estradiol can inhibit the activation of microglias, reducing the expression of iNOS, thereby reducing the synthesis of NO nerve toxic substances.3. NO is nerotoxic.4. The test of RGCs’TUNEL showed thatβ-estradiol can reduce the apoptosis of RGCs, so it can protect.5. The expression of CD11b and iNOS of the three groups are both assumed linear correlation with the expression of RGCs’TNUEL,and confirmed that microglia cells played a regulatory role to apoptosis of RGCs in the high intraocular pressure.更多还原