本文選題：青光眼　切入點(diǎn)：雌激素　出處：《河北醫(yī)科大學(xué)》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(jī)制尚不清楚,其中青光眼免疫學(xué)發(fā)病機(jī)制逐漸引起人們的關(guān)注。視網(wǎng)膜小膠質(zhì)細(xì)胞作為中樞神經(jīng)系統(tǒng)最具代表性的免疫細(xì)胞已成為研究熱點(diǎn),其視神經(jīng)保護(hù)與毒性的雙重作用歸因于它對(duì)一系列病變做出反應(yīng)后所能合成和分泌的物質(zhì)不同,因此針對(duì)視網(wǎng)膜小膠質(zhì)細(xì)胞雙重作用的藥物研究成為治療青光眼的切入點(diǎn)。有研究表明雌激素具有免疫細(xì)胞凋亡的調(diào)節(jié)作用,亦有視神經(jīng)保護(hù)作用,故本實(shí)驗(yàn)通過建立大鼠急性高眼壓模型,觀察雌激素對(duì)視網(wǎng)膜小膠質(zhì)細(xì)胞的活化、誘導(dǎo)型一氧化碳合酶(iNOS)的表達(dá)影響及對(duì)視神經(jīng)節(jié)細(xì)胞的保護(hù),為今后治療青光眼提供實(shí)驗(yàn)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(nèi)源性雌激素對(duì)實(shí)驗(yàn)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(duì)照眼(以下統(tǒng)稱為自身對(duì)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(shí)驗(yàn)中突然卒死及不符合標(biāo)準(zhǔn)的眼球,每組各保留10只眼球固定后行石蠟切片,應(yīng)用免疫組化技術(shù)分析視網(wǎng)膜小膠質(zhì)細(xì)胞的表達(dá)(細(xì)胞表面標(biāo)記物為CD11b)及其iNOS的表達(dá),以及視神經(jīng)節(jié)細(xì)胞的凋亡。計(jì)算機(jī)圖像分析軟件對(duì)圖像中的陽性反應(yīng)部位進(jìn)行光密度分析,三者半定量測(cè)定結(jié)果均以均數(shù)±標(biāo)準(zhǔn)差(Mean±SD)表示,應(yīng)用SPSS統(tǒng)計(jì)分析軟件進(jìn)行統(tǒng)計(jì)學(xué)分析 結(jié)果:1.組織病理學(xué)變化: 1.1CD11b陽性小膠質(zhì)細(xì)胞:Ⅰ組可見小膠質(zhì)細(xì)胞淡染,呈棕黃色、分枝狀,胞體小,具有伸向各個(gè)方向的細(xì)小突起。Ⅱ、Ⅲ組模型眼活化的小膠質(zhì)細(xì)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(xì)胞及細(xì)胞碎片周圍。 1.2視神經(jīng)節(jié)細(xì)胞:Ⅰ組視網(wǎng)膜神經(jīng)節(jié)細(xì)胞呈單層排列,無陽性細(xì)胞。Ⅱ、Ⅲ組的自身對(duì)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(xì)胞數(shù)目明顯減少,視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層呈空泡樣改變,部分細(xì)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(xì)胞數(shù)目減少,形態(tài)大致正常,神經(jīng)節(jié)細(xì)胞層結(jié)構(gòu)基本正常。 2.小膠質(zhì)細(xì)胞免疫組織化學(xué)分析(即小膠質(zhì)細(xì)胞的活化及iNOS的表達(dá)):3組CD11b陽性細(xì)胞數(shù)均可檢測(cè)到,Ⅰ組未見到iNOS陽性細(xì)胞,Ⅱ、Ⅲ組模型眼CD11b陽性細(xì)胞及iNOS陽性細(xì)胞增多,均高于Ⅰ組及自身對(duì)照眼(P0.05)。Ⅱ組模型眼染色CD11b陽性細(xì)胞及iNOS陽性細(xì)胞數(shù)少于Ⅲ組模型眼陽性細(xì)胞數(shù)(P0.05)。 3.視神經(jīng)節(jié)細(xì)胞TUNEL原位凋亡監(jiān)測(cè):染色陽性的視網(wǎng)膜節(jié)細(xì)胞(RGCs)在3組動(dòng)物眼球上均可檢測(cè)到,Ⅰ組及Ⅱ、Ⅲ組的自身對(duì)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(xì)胞數(shù)均高于Ⅰ組及自身對(duì)照眼(P0.05)。Ⅱ組模型眼TUNEL染色陽性細(xì)胞數(shù)少于Ⅲ組模型眼陽性細(xì)胞數(shù)(P0.05)。同時(shí)證實(shí)小膠質(zhì)細(xì)胞的活化及其iNOS的表達(dá)均與節(jié)細(xì)胞凋亡指數(shù)呈正相關(guān)。 結(jié)論: 1.通過分析Ⅱ、Ⅲ組模型眼與Ⅰ組及自身對(duì)照眼CD11b及iNOS的表達(dá)(P0.05),證實(shí)高眼壓下小膠質(zhì)細(xì)胞活化,并表達(dá)iNOS。 2.Ⅱ、Ⅲ組模型眼的CD11b及iNOS表達(dá)差異有統(tǒng)計(jì)學(xué)意義(P0.05),表明雌激素能夠抑制小膠質(zhì)細(xì)胞的活化,減少iNOS的表達(dá),從而減少合成NO神經(jīng)毒性物質(zhì)。 3.NO具有神經(jīng)毒性,。 4.RGCs的TUNEL檢測(cè)表明雌激素能夠減少節(jié)細(xì)胞的凋亡,從而保護(hù)視神經(jīng)節(jié)細(xì)胞。 5.3組CD11b的表達(dá)、iNOS表達(dá)均與TUNEL的表達(dá)呈直線正相關(guān),說明在高眼壓下小膠質(zhì)細(xì)胞對(duì)節(jié)細(xì)胞的凋亡起到了調(diào)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.
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