雌激素对大鼠急性高眼压模型中小胶质细胞的影响

发布时间：2018-03-27 09:53

  本文选题：青光眼　切入点：雌激素　出处：《河北医科大学》2010年硕士论文

【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(P0.05)。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(P0.05)。 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(P0.05)。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(P0.05)。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(P0.05),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(P0.05),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。
[Abstract]:Objective: glaucoma is one of the most common clinical cause of blindness, the pathogenesis is not clear, the pathogenesis of glaucoma immunology has aroused people's concern. Retinal microglia in the central nervous system as the most representative of the immune cells has become a hot research topic, the dual role of neuroprotection and toxicity due to synthesis and secretion different substances in response to a series of pathological changes, so the study drugs for the dual role of retinal microglia become the starting point for treatment of glaucoma. Studies have shown that estrogen can regulate immune cell apoptosis, but also protect the optic nerve, so this study established a rat model of acute high intraocular pressure, activation of observation effect of estrogen on retinal microglia, inducible nitric oxide synthase (iNOS) expression and effect of optic ganglion cells protection, for this The experimental basis for the treatment of glaucoma after treatment is provided.
Methods: 35 SD rats (male with male rats is designed to minimize the effect of endogenous estrogen on the experiment), no eye, weight 200 + 20 g, divided into 3 groups, group I: normal group of 5 rats (10 eyes), more than 30 were using anterior chamber pressure perfusion method will be made of the left eye acute high intraocular pressure model (hereinafter referred to as the model for their own eyes), eye to eye (hereinafter referred to as the control group), then the 30 were randomly divided into group II: estrogen intraperitoneal injection group 15 and group III: disinfection of olive oil injection group 15. Finally abandoned to the experiment of sudden death and sudden in accordance with the eye, each retain 10 eyes were fixed in paraffin sections, the expression of immunohistochemical analysis of retinal microglia (cell surface markers for CD11b and iNOS) expression, and apoptosis of retinal ganglion cells. The positive reaction of computer image analysis software for image. The optical density analysis was carried out. The results of the semi quantitative determination of the three subjects were all expressed in the mean number of standard deviation (Mean + SD). The statistical analysis software of SPSS was used for statistical analysis.
Results: 1. the changes of histopathology:
1.1CD11b positive microglia: group I showed small glial cells stained, brownish yellow, branched, small cell body, with thin projections extending in all directions. II, III group model eye number of activated microglia increase, neurites shorter, thicker, cell body size, round, around the festival cells and cell debris and gathered from apoptosis.
1.2 retinal ganglion cell: group I retinal ganglion cells formed a monolayer, no positive cells. II, III group itself in control and I group. The same model II, III group vision mirror showed significantly reduced the number of retinal ganglion cells, retinal ganglion cell layer showed vacuolar changes, cell nuclear dissolution, nuclear light staining, the cytoplasm stained lightly. Group II model retinal ganglion cells decreased the number, morphology are normal, ganglion cell layer structure is normal.
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