艾地苯醌对缺血再灌注视网膜损伤的作用观察

发布时间：2018-10-11 19:58

【摘要】：背景及目的视网膜缺血再灌注损伤(Retina ischemia-reperfusion injury,RIRI)是临床上主要致盲性眼病共同的病理生理过程,如青光眼、视网膜中央动脉阻塞、糖尿病性视网膜病变等。其可通过诱导视网膜神经节细胞(RGCs)凋亡导致失明,其中线粒体功能障碍引起的氧化应激及凋亡相关蛋白的表达和释放是重要机制之一。辅酶Q10是线粒体电子传递链中必不可少的辅助因子和强效的抗氧化剂,在神经退行性疾病中对神经元细胞的氧化应激损伤有明显的保护作用。艾地苯醌是一种已经应用于临床的线粒体功能靶向药物,与辅酶Q10有类似的醌环结构,但较辅酶Q10有更强的水溶性,易于被人体所吸收。艾地苯醌在治疗脑血管疾病以及遗传性线粒体功能障碍性疾病中的抗氧化和神经保护作用已经得到证实。本研究通过提高前房灌注压制作大鼠缺血再灌注模型,艾地苯醌干预并检测细胞色素C(Cytochrome C)、Caspase-3表达及RGCs计数,来探索艾地苯醌在RIRI中的作用,从而为治疗青光眼、糖尿病性视网膜病变等RIRI疾病提供新的治疗思路。方法健康雄性SD大鼠随机分为3组:空白对照组,实验对照组,实验治疗组。HE染色观察再灌注12h,24h,48h,72h视网膜组织形态改变并利用免疫组织化学方法检测caspase-3表达。Western-blot方法检测再灌注24h Cytochrome C表达。利用免疫组织荧光方法检测再灌注7d后,Thy1 1标记RGCs并计数。SPSS21.0软件进行数据分析,定量结果用x±s表示,界定P0.05为差异具有统计学意义。结果1.HE观察大鼠视网膜形态及结构改变:空白对照组视网膜组织结构清晰;实验对照组视网膜水肿,结构紊乱,神经节细胞层细胞数量明显减少;与实验对照组相比,实验治疗组大鼠视网膜结构较完整,水肿程度轻,神经节细胞层细胞数量也较多。2.免疫组化:空白对照组视网膜各层caspase-3几乎无表达;实验对照组Caspase-3表达明显增加;实验治疗组较实验对照组Caspase-3表达减少。Caspase-3表达主要集中在神经节细胞层及内核层,随时间变化呈现先增高、后降低趋势,24h组Caspase-3表达量最高。各时间亚组差异有统计学意义(P0.05)。3.Western-blot:空白对照组Cytochrome C表达微弱,实验对照组Cytochrome C表达迅速增加,实验治疗组较实验对照组Cytochrome C表达减少。经统计学分析差异有统计学意义(P0.05)。4.免疫荧光:空白对照组RGCs计数结果为15.90±1.20个/视野;实验对照组RGCs明显减少,计数结果为7.70±2.31个/视野;实验治疗组RGCs较实验对照组增多,计数结果为10.70±2.11个/视野。经统计学分析差异有统计学意义(P0.05)。结论1.提高前房灌注压制作视网膜缺血再灌注模型可导致大鼠视网膜Cytochrome C、Caspase-3表达增多,RGCs存活数目减少。2.艾地苯醌干预可减少大鼠视网膜Cytochrome C、Caspase-3表达,增加RGCs存活数目。其神经保护作用可能通过稳定线粒体氧化呼吸链来实现。
[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 卤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 卤1.20 / visual field, the RGCs count in the experimental control group was 7.70 卤2.31 / visual field, and the RGCs count in the experimental treatment group was 10.70 卤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.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R774.1

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