本文選題：青光眼　切入點：雌激素　出處：《河北醫(yī)科大學》2010年碩士論文

【摘要】： 目的:青光眼是臨床最常見致盲眼病之一,發(fā)病機制尚不清楚,其中青光眼免疫學發(fā)病機制逐漸引起人們的關注。視網(wǎng)膜小膠質細胞作為中樞神經(jīng)系統(tǒng)最具代表性的免疫細胞已成為研究熱點,其視神經(jīng)保護與毒性的雙重作用歸因于它對一系列病變做出反應后所能合成和分泌的物質不同,因此針對視網(wǎng)膜小膠質細胞雙重作用的藥物研究成為治療青光眼的切入點。有研究表明雌激素具有免疫細胞凋亡的調節(jié)作用,亦有視神經(jīng)保護作用,故本實驗通過建立大鼠急性高眼壓模型,觀察雌激素對視網(wǎng)膜小膠質細胞的活化、誘導型一氧化碳合酶(iNOS)的表達影響及對視神經(jīng)節(jié)細胞的保護,為今后治療青光眼提供實驗依據(jù)。 方法:SD大鼠35只雄性(全部采用雄性大鼠的目的是盡量減少內源性雌激素對實驗的影響),無眼疾,體重200±20 g,分3組,Ⅰ組即正常組5只(10眼),余30只均采用前房加壓灌注法將左眼制成急性高眼壓模型(以下統(tǒng)稱模型眼),右眼為自身對照眼(以下統(tǒng)稱為自身對照眼),再將這30只分為Ⅱ組即雌激素腹腔注射組15只和Ⅲ組即消毒橄欖油注射組15只。最終棄去實驗中突然卒死及不符合標準的眼球,每組各保留10只眼球固定后行石蠟切片,應用免疫組化技術分析視網(wǎng)膜小膠質細胞的表達(細胞表面標記物為CD11b)及其iNOS的表達,以及視神經(jīng)節(jié)細胞的凋亡。計算機圖像分析軟件對圖像中的陽性反應部位進行光密度分析,三者半定量測定結果均以均數(shù)±標準差(Mean±SD)表示,應用SPSS統(tǒng)計分析軟件進行統(tǒng)計學分析 結果:1.組織病理學變化: 1.1CD11b陽性小膠質細胞:Ⅰ組可見小膠質細胞淡染,呈棕黃色、分枝狀,胞體小,具有伸向各個方向的細小突起。Ⅱ、Ⅲ組模型眼活化的小膠質細胞數(shù)量增加,突起變短、變粗,胞體增大、變圓,并聚集于凋亡的節(jié)細胞及細胞碎片周圍。 1.2視神經(jīng)節(jié)細胞:Ⅰ組視網(wǎng)膜神經(jīng)節(jié)細胞呈單層排列,無陽性細胞。Ⅱ、Ⅲ組的自身對照眼與I組相同。Ⅱ、Ⅲ組的模型眼光鏡下可見視網(wǎng)膜神經(jīng)節(jié)細胞數(shù)目明顯減少,視網(wǎng)膜神經(jīng)節(jié)細胞層呈空泡樣改變,部分細胞出現(xiàn)核溶解、核染色淺淡、胞漿染色淺淡。Ⅱ組模型眼視網(wǎng)膜神經(jīng)節(jié)細胞數(shù)目減少,形態(tài)大致正常,神經(jīng)節(jié)細胞層結構基本正常。 2.小膠質細胞免疫組織化學分析(即小膠質細胞的活化及iNOS的表達):3組CD11b陽性細胞數(shù)均可檢測到,Ⅰ組未見到iNOS陽性細胞,Ⅱ、Ⅲ組模型眼CD11b陽性細胞及iNOS陽性細胞增多,均高于Ⅰ組及自身對照眼(P0.05)。Ⅱ組模型眼染色CD11b陽性細胞及iNOS陽性細胞數(shù)少于Ⅲ組模型眼陽性細胞數(shù)(P0.05)。 3.視神經(jīng)節(jié)細胞TUNEL原位凋亡監(jiān)測:染色陽性的視網(wǎng)膜節(jié)細胞(RGCs)在3組動物眼球上均可檢測到,Ⅰ組及Ⅱ、Ⅲ組的自身對照眼TUNEL染色陽性的RGCs數(shù)最少。Ⅱ、Ⅲ組模型眼上TUNEL染色陽性的RGCs稍多,陽性細胞數(shù)均高于Ⅰ組及自身對照眼(P0.05)。Ⅱ組模型眼TUNEL染色陽性細胞數(shù)少于Ⅲ組模型眼陽性細胞數(shù)(P0.05)。同時證實小膠質細胞的活化及其iNOS的表達均與節(jié)細胞凋亡指數(shù)呈正相關。 結論: 1.通過分析Ⅱ、Ⅲ組模型眼與Ⅰ組及自身對照眼CD11b及iNOS的表達(P0.05),證實高眼壓下小膠質細胞活化,并表達iNOS。 2.Ⅱ、Ⅲ組模型眼的CD11b及iNOS表達差異有統(tǒng)計學意義(P0.05),表明雌激素能夠抑制小膠質細胞的活化,減少iNOS的表達,從而減少合成NO神經(jīng)毒性物質。 3.NO具有神經(jīng)毒性,。 4.RGCs的TUNEL檢測表明雌激素能夠減少節(jié)細胞的凋亡,從而保護視神經(jīng)節(jié)細胞。 5.3組CD11b的表達、iNOS表達均與TUNEL的表達呈直線正相關,說明在高眼壓下小膠質細胞對節(jié)細胞的凋亡起到了調節(jié)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.
2.灝忚兌璐ㄧ粏鑳?yōu)鍏嶇柅缁劸l囧寲瀛﹀垎鏋，

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