【摘要】：背景及目的視網(wǎng)膜缺血再灌注損傷(Retina ischemia-reperfusion injury,RIRI)是臨床上主要致盲性眼病共同的病理生理過程,如青光眼、視網(wǎng)膜中央動脈阻塞、糖尿病性視網(wǎng)膜病變等。其可通過誘導視網(wǎng)膜神經(jīng)節(jié)細胞(RGCs)凋亡導致失明,其中線粒體功能障礙引起的氧化應激及凋亡相關蛋白的表達和釋放是重要機制之一。輔酶Q10是線粒體電子傳遞鏈中必不可少的輔助因子和強效的抗氧化劑,在神經(jīng)退行性疾病中對神經(jīng)元細胞的氧化應激損傷有明顯的保護作用。艾地苯醌是一種已經(jīng)應用于臨床的線粒體功能靶向藥物,與輔酶Q10有類似的醌環(huán)結構,但較輔酶Q10有更強的水溶性,易于被人體所吸收。艾地苯醌在治療腦血管疾病以及遺傳性線粒體功能障礙性疾病中的抗氧化和神經(jīng)保護作用已經(jīng)得到證實。本研究通過提高前房灌注壓制作大鼠缺血再灌注模型,艾地苯醌干預并檢測細胞色素C(Cytochrome C)、Caspase-3表達及RGCs計數(shù),來探索艾地苯醌在RIRI中的作用,從而為治療青光眼、糖尿病性視網(wǎng)膜病變等RIRI疾病提供新的治療思路。方法健康雄性SD大鼠隨機分為3組:空白對照組,實驗對照組,實驗治療組。HE染色觀察再灌注12h,24h,48h,72h視網(wǎng)膜組織形態(tài)改變并利用免疫組織化學方法檢測caspase-3表達。Western-blot方法檢測再灌注24h Cytochrome C表達。利用免疫組織熒光方法檢測再灌注7d后,Thy1 1標記RGCs并計數(shù)。SPSS21.0軟件進行數(shù)據(jù)分析,定量結果用x±s表示,界定P0.05為差異具有統(tǒng)計學意義。結果1.HE觀察大鼠視網(wǎng)膜形態(tài)及結構改變:空白對照組視網(wǎng)膜組織結構清晰;實驗對照組視網(wǎng)膜水腫,結構紊亂,神經(jīng)節(jié)細胞層細胞數(shù)量明顯減少;與實驗對照組相比,實驗治療組大鼠視網(wǎng)膜結構較完整,水腫程度輕,神經(jīng)節(jié)細胞層細胞數(shù)量也較多。2.免疫組化:空白對照組視網(wǎng)膜各層caspase-3幾乎無表達;實驗對照組Caspase-3表達明顯增加;實驗治療組較實驗對照組Caspase-3表達減少。Caspase-3表達主要集中在神經(jīng)節(jié)細胞層及內核層,隨時間變化呈現(xiàn)先增高、后降低趨勢,24h組Caspase-3表達量最高。各時間亞組差異有統(tǒng)計學意義(P0.05)。3.Western-blot:空白對照組Cytochrome C表達微弱,實驗對照組Cytochrome C表達迅速增加,實驗治療組較實驗對照組Cytochrome C表達減少。經(jīng)統(tǒng)計學分析差異有統(tǒng)計學意義(P0.05)。4.免疫熒光:空白對照組RGCs計數(shù)結果為15.90±1.20個/視野;實驗對照組RGCs明顯減少,計數(shù)結果為7.70±2.31個/視野;實驗治療組RGCs較實驗對照組增多,計數(shù)結果為10.70±2.11個/視野。經(jīng)統(tǒng)計學分析差異有統(tǒng)計學意義(P0.05)。結論1.提高前房灌注壓制作視網(wǎng)膜缺血再灌注模型可導致大鼠視網(wǎng)膜Cytochrome C、Caspase-3表達增多,RGCs存活數(shù)目減少。2.艾地苯醌干預可減少大鼠視網(wǎng)膜Cytochrome C、Caspase-3表達,增加RGCs存活數(shù)目。其神經(jīng)保護作用可能通過穩(wěn)定線粒體氧化呼吸鏈來實現(xiàn)。
[Abstract]:Background and objective Retinal ischemia-reperfusion injury (Retina ischemia-reperfusion injury,RIRI) is a common pathophysiological process of primary blinding ophthalmopathy, such as glaucoma, central retinal artery occlusion, diabetic retinopathy and so on. It can induce blindness by inducing apoptosis of retinal ganglion cells (RGCs). Oxidative stress induced by mitochondrial dysfunction and the expression and release of apoptosis-related proteins are one of the important mechanisms. Coenzyme Q10 is an essential cofactor and potent antioxidant in mitochondrial electron transport chain. It can protect neurons from oxidative stress injury in neurodegenerative diseases. Ai dibenzoquinone is a kind of mitochondrial functional targeting drug which has been used in clinic. It has a similar quinone ring structure to coenzyme Q10, but it is more water-soluble than coenzyme Q10 and is easily absorbed by human body. The antioxidant and neuroprotective effects of Adibenzoquinone in the treatment of cerebrovascular diseases and hereditary mitochondrial dysfunction have been confirmed. The purpose of this study was to investigate the role of Ai dibenzoquinone in the treatment of glaucoma by increasing the pressure of anterior chamber perfusion to make the model of ischemia reperfusion in rats, and to detect the expression of cytochrome C (Cytochrome C), Caspase-3 and the count of RGCs, and to investigate the role of Ai dibenzoquinone in the treatment of glaucoma. Diabetic retinopathy and other RIRI diseases provide a new approach to the treatment. Methods healthy male SD rats were randomly divided into three groups: blank control group and experimental control group. In the experimental treatment group, HE staining was used to observe the morphological changes of retinal tissue and the expression of caspase-3 was detected by immunohistochemical method, and the expression of Cytochrome C at 24 h after reperfusion was detected by Western-blot method. After 7 days of reperfusion, RGCs was labeled with Thy1 1 and counted. SPSS21.0 software was used to analyze the data. The quantitative results were expressed as x 鹵s, and the difference was statistically significant. Results the morphological and structural changes of rat retina were observed by 1.HE: the retinal tissue structure was clear in the blank control group, the retinal edema, the structure disorder and the number of ganglion cell layer cells in the experimental control group were significantly decreased, compared with the experimental control group. In the experimental group, the retinal structure was relatively complete, the degree of edema was light, and the number of ganglion cell layer cells was more than that of the control group. Immunohistochemistry: there was almost no expression of caspase-3 in all layers of retina in blank control group; Caspase-3 expression in experimental control group was significantly increased; Caspase-3 expression in experimental treatment group was lower than that in experimental control group. The expression of Caspase-3 was mainly in ganglion cell layer and nuclear layer. With the change of time, the expression of Caspase-3 increased first, then decreased, and the expression of Caspase-3 was the highest in 24 h group. Western-blot: the expression of Cytochrome C in blank control group was weak, the expression of Cytochrome C in experimental control group was increasing rapidly, and the expression of Cytochrome C in experimental group was lower than that in experimental control group. The difference was statistically significant (P0.05). 4. Immunofluorescence: the RGCs count in the blank control group was 15.90 鹵1.20 / visual field, the RGCs count in the experimental control group was 7.70 鹵2.31 / visual field, and the RGCs count in the experimental treatment group was 10.70 鹵2.11 / visual field as compared with that in the experimental control group. The difference was statistically significant (P0.05). Conclusion 1. Increasing the pressure of anterior chamber perfusion to make retinal ischemia-reperfusion model could increase the expression of Caspase-3 and decrease the number of RGCs survival in rat retina. Adibenzoquinone could decrease the expression of Caspase-3 and increase the number of RGCs survival in rat retina. Its neuroprotective effect may be achieved by stabilizing mitochondrial oxidative respiratory chain.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R774.1

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