盐酸法舒地尔对兔视网膜缺血再灌注损伤神经细胞凋亡及Bcl-2、Caspase-3蛋白表达的影响

发布时间：2018-04-27 19:39

本文选题：盐酸法舒地尔 + 视网膜缺血再灌注损伤　；参考：《河北联合大学》2014年硕士论文

【摘要】：目的建立一种能模拟人类RIRI特征的、可靠的动物模型，经兔耳缘静脉注射盐酸法舒地尔后，并分别通过HE染色法观察视网膜形态学变化、TUNEL法检测神经细胞凋亡的变化及免疫组化法检测凋亡基因Bcl-2、Caspase-3蛋白表达的变化及盐酸法舒地尔对其的影响，从而进一步探讨盐酸法舒地尔对RIRI的神经保护及作用机制，为RIRI的临床治疗提供理论依据。材料与方法1实验动物分组：本研究选取54只健康雄性日本纯种大耳白兔作为研究对象，按照平行对照原则随机分为3组，其分别为：正常组（6只）、损伤组（24只）、治疗组（24只），其中损伤组和治疗组又按照不同的再灌注时间点（6h、12h、24h、48h、72h、120h）分为6组。2建立RIRI动物模型：损伤组和治疗组均采取前房灌注加压法造成视网膜缺血，并在缺血1h后，将眼内压迅速降至正常水平，从而制作成RIRI动物模型。3给药途径及方式：三组给药途径均为耳缘静脉注射，治疗组注射盐酸法舒地尔剂量为10mg/kg，正常组和损伤组则注射等剂量的平衡盐溶液。4标本制作及方法：各组分别在再灌注后6h、12h、24h、48h、72h及120h处死动物并迅速摘除眼球，对不同时间段的视网膜组织分别通过HE染色法观察视网膜形态学变化，并通过TUNEL法检测神经细胞凋亡的变化和通过免疫组化法检测凋亡基因Bcl-2、Caspase-3蛋白表达的变化。结果1视网膜形态学的变化：治疗组的视网膜损伤程度明显轻于损伤组。2视网膜神经细胞凋亡的变化：在6h、12h、24h、48h、72h时间点，三组间同一时间点凋亡细胞计数结果两两比较均具有显著统计学意义（P＜0.01），而120h时各组间比较无明显统计学意义（P＞0.05）。损伤组：各个时间点上凋亡细胞计数比较有显著统计学意义（F值=13.06，P＜0.01）；治疗组：各个时间点上凋亡细胞计数比较有显著统计学意义（F值=22.54，P＜0.01）。3凋亡基因Bcl-2蛋白的表达：在6h、12h、24h、48h、72h时间点，三组间同一时间点Bcl-2蛋白表达两两比较均具有显著统计学意义（P＜0.01），而120h时各组间比较无明显统计学意义（P＞0.05）。损伤组：各个时间点Bcl-2蛋白表达比较有显著统计学意义（F值=15.14，P＜0.01）；治疗组：各个时间点之间Bcl-2蛋白表达比较有显著统计学意义（F值=17.06，P＜0.01）。4凋亡基因Caspase-3蛋白的表达：在6h、12h、24h、48h、72h、120h时间点，三组间同一时间点Caspase-3蛋白表达两两比较均具有显著统计学意义（P＜0.01）。损伤组：各个时间点之间阳性细胞计数差异有显著统计学意义(F值=65.860，P＜0.01）；治疗组：各个时间点之间阳性细胞计数差异有显著统计学意义（F值=276.550，P＜0.01）。结论1盐酸法舒地尔能减轻视网膜缺血再灌注损伤，在维持视网膜的形态及功能中起着重要的作用；2盐酸法舒地尔能抑制视网膜神经细胞的凋亡，其作用机制可能与提高Bcl-2及降低Caspase-3表达有关，从而在视网膜缺血再灌注损伤中对视网膜神经细胞起到明显的保护作用。
[Abstract]:Objective to establish a reliable animal model which can mimic the characteristics of human RIRI. The morphological changes of retina were observed by HE staining and apoptosis of neurons was detected by Tunel method and the expression of apoptotic gene Bcl-2Caspase-3 was detected by immunohistochemical method and the effect of fasudil hydrochloride on it was also observed. To further explore the neuroprotection and mechanism of fasudil hydrochloride on RIRI and provide theoretical basis for the clinical treatment of RIRI. Materials and methods 1 Experimental animals were divided into three groups: 54 healthy male purebred Japanese white rabbits were randomly divided into 3 groups according to the principle of parallel control. They were: normal group (n = 6), injury group (n = 24) and treatment group (n = 24). According to different reperfusion time points, the injury group and the treatment group were divided into 6 groups to establish RIRI animal model: the injury group and the treatment group adopted anterior chamber. The retinal ischemia is caused by perfusion and compression. After 1 hour of ischemia, intraocular pressure was rapidly reduced to normal level, and then RIRI animal model was established. 3 administration route and method: all three groups were injected via ear vein. In the treatment group, the dose of fasudil hydrochloride was 10 mg / kg, the control group and the injury group were injected with the same dose of balanced salt solution of 4. 4 samples and methods: the animals were killed at 6 h, 12 h, 24 h, 48 h, 72 h and 120 h after reperfusion, and their eyeballs were removed quickly. The morphological changes of retina were observed by HE staining, the changes of neuronal apoptosis and the expression of apoptotic gene Bcl-2Caspase-3 were detected by TUNEL method and immunohistochemistry method respectively. Results 1 changes of retinal morphology: the degree of retinal injury in the treatment group was significantly less than that in the injured group (group .2): at the time point of 6h, 12h, 24h, 48h and 72h, the degree of retinal injury in the treatment group was significantly less than that in the injured group (group .2). The number of apoptotic cells in the three groups at the same time point was significantly different (P < 0.01), but there was no significant difference between the three groups at 120 h (P > 0.05). In the injury group, the number of apoptotic cells at each time point was significantly higher than that in the control group (P < 0.01), and in the treatment group, the number of apoptotic cells was significantly higher than that in the control group (P < 0.01), and the expression of apoptotic gene Bcl-2 protein was significantly higher in the treatment group than that in the treatment group at 12 h, 24 h, 48 h, 72 h, respectively. The expression of Bcl-2 protein in the three groups at the same time point was significantly higher than that in the control group (P < 0.01), but there was no significant difference between the three groups at 120h (P > 0.05). In the injury group, the expression of Bcl-2 protein was significantly higher than that in the control group (P < 0.01), and in the treatment group, the expression of Bcl-2 protein was significantly higher than that in the control group (P < 0.01). The expression of Caspase-3 protein of apoptotic gene was significantly higher in the treatment group than that in the control group at 12 h, 24 h, 48 h, 72 h and 120 h, respectively. The expression of Caspase-3 protein in the three groups was significantly different at the same time point (P < 0.01). In the injury group, there was significant difference in the number of positive cells between different time points (P < 0.01), and in the treatment group, there was significant difference in the number of positive cells between different time points (P < 0.01). Conclusion 1 Faxudil hydrochloride can attenuate retinal ischemia-reperfusion injury and play an important role in the maintenance of retinal morphology and function. Second, fasudil hydrochloride can inhibit the apoptosis of retinal nerve cells. The mechanism may be related to the increase of Bcl-2 and the decrease of Caspase-3 expression, which may play an important role in protecting retinal nerve cells from retinal ischemia reperfusion injury.
【学位授予单位】：河北联合大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965

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