小鼠局灶性脑缺血再灌注模型建立及二氢杨梅素干预研究

发布时间：2018-04-27 19:26

本文选题：小鼠 + 脑缺血再灌注损伤　；参考：《广西医科大学》2014年硕士论文

【摘要】：脑缺血再灌注损伤是一个多种致病因子参与的复杂病理生理过程，该损伤的分子生物学机制至今尚未能够彻底阐明；缺血性脑血管病严重威胁着人类的健康，现代医学对于缺血性脑卒中的常规疗法疗效不佳，毒副作用大，急需开发低毒高效的药物。本文主要研究了小鼠局灶性脑缺血再灌注模型的改良方法以及二氢杨梅素对脑缺血再灌注损伤的保护作用，并证实了此种保护作用与其抗氧化功效相关。试图为拓展DMY在临床上对于缺血性脑卒中的应用、开发低毒高效的抗卒中药物，奠定良好的模型基础，并提供实验数据支持。第一部分小鼠局灶性脑缺血再灌注模型的建立与评价目的：建立小鼠局灶性脑缺血再灌注(I/R)模型并予以评价。方法：雄性昆明小鼠60只随机分为假手术组30只，I/R模型组30只。改良线栓法制备小鼠大脑中动脉阻塞/再灌注（MCAO/R）模型。通过神经行为学评分、氯化三苯基四氮唑（TTC）染色法测定脑梗死比（%）、干湿重法测定脑含水量（%）及HE染色观察脑组织病理改变评价模型可靠性。结果：模型存活率为83.33%，总成功率为76.67％。假手术组小鼠神经行为学评分为（0±0）分，脑梗死比为（0±0）％，脑含水量（77.29±0.45）%。I/R模型组小鼠神经行为学评分为（2.42±0.63）分，脑梗死比为（23.03±3.42）％，脑含水量（83.18±1.65）%，均较假手术组明显增高（P0.01）；病理检查出现典型脑梗死病理改变。结论:改良线栓法成功建立简便、可靠的小鼠局灶性脑缺血再灌注模型。第二部分二氢杨梅素对小鼠局灶性脑缺血再灌注损伤的保护作用目的：研究二氢杨梅素(Dihydromyricetin，DMY)对局灶性脑缺血再灌注(I/R)损伤的保护作用。方法：雄性昆明种小鼠随机分为4组，，即假手术组、I/R模型组及DMY高、低剂量组。模型组和DMY组采用线栓法建立左侧大脑中动脉阻塞(middle cerebral artery occlusion, MCAO)3h/再灌注24h局灶性脑缺血再灌注损伤模型；假手术组仅结扎左侧颈总动脉，不插入线栓。DMY高、低剂量组分别在造模前10d持续灌胃DMY500、250mg kg-1，并在缺血前1h、再灌注前1h及再灌注后6h、12h分别灌胃1次；缺血再灌注组和假手术组则同期灌胃等体积0.5%羧甲基纤维素钠。再灌注24后，观察小鼠神经功能缺损症状评分，取脑行TTC染色测定脑梗死比（%）、干湿重法测定脑含水量（%），HE染色观察脑组织病理学变化，计数皮层半暗带区神经元数目。结果：与模型组比较，DMY250mg kg-1500mg kg-1组均能降低脑缺血损伤后脑梗死比；DMY500mg kg-1组还可降低神经功能缺损评分和脑含水量，改善脑组织病理形态，增加皮层半暗带区神经元数量（P 0.05或P 0.01）。结论：DMY对小鼠局灶性脑缺血再灌注损伤具有一定的保护作用。第三部分二氢杨梅素对小鼠局灶性脑缺血再灌注的抗氧化保护机制研究目的：研究二氢杨梅素（DMY）对局灶性脑缺血再灌注损伤的抗氧化保护机制。方法：雄性昆明种小鼠40只随机分为4组，即假手术组、I/R模型组及DMY高、低剂量组，每组10只。MCAO模型建立和DMY给药方法同“第二部分”。缺血3h再灌注24h后，分别测定各组小鼠缺血侧脑组织中MDA含量和SOD、GSH-PX的活力。结果：与模型组比较，DMY500mg kg-1组能明显降低脑缺血损伤后脑组织MDA水平（P 0.01），增加I/R损伤后脑组织中t-SOD和GSH-PX的水平（P 0.01）。结论：DMY保护小鼠局灶性脑I/R损伤与其抗氧化作用有关。
[Abstract]:Cerebral ischemia-reperfusion injury is a complex pathophysiological process involving a variety of pathogenic factors. The molecular biological mechanism of this injury has not yet been fully elucidated. Ischemic cerebrovascular disease is a serious threat to human health. The conventional therapy for ischemic stroke is not effective in modern medicine, toxic and side effects, and need to be developed in urgent need of low development. A drug that is highly toxic and efficient.
This article mainly studied the improved method of focal cerebral ischemia reperfusion model in mice and the protective effect of two hydrogen myricetin on cerebral ischemia reperfusion injury, and confirmed that this protective effect is related to its antioxidation effect. The purpose of this study is to develop the clinical application of DMY for ischemic stroke and develop low toxic and efficient anti stroke drugs. Lay a good foundation for the model and provide experimental data support.
Part one establishment and evaluation of focal cerebral ischemia-reperfusion model in mice
Objective: to establish a mouse model of focal cerebral ischemia-reperfusion (I/R) and evaluate it.
Methods: 60 male Kunming mice were randomly divided into 30 sham operation group and 30 I/R model group. The modified line embolus method was used to prepare the middle cerebral artery occlusion / reperfusion (MCAO/R) model in mice. The cerebral infarction ratio (%) was measured by the neurobehavioral score, three phenyl tetrazolium chloride (TTC) staining method was used to determine the brain water content (%) and the HE staining was used to observe the brain group. The evaluation model of pathological changes was reliable.
Results: the survival rate of the model was 83.33%, the total success rate was (0 + 0), cerebral infarction ratio (0 + 0)%, cerebral water content (77.29 + 0.45) model group (2.42 + 0.63), cerebral infarction ratio (23.03 + 3.42)%, cerebral water content (23.03 + 0)%, and cerebral water content (83.18 + 1.65)%, compared with sham operation group (0 + 0)% (0 + 0)% of the mice in 76.67%. sham operation group. Obviously increased (P0.01); pathological examination showed typical pathological changes of cerebral infarction.
Conclusion: a simple and reliable model of focal cerebral ischemia-reperfusion in mice was successfully established by modified thread embolism.
The second part: protective effect of two hydrogen myricetin on focal cerebral ischemia-reperfusion injury in mice
Objective: To study the protective effect of two Dihydromyricetin (DMY) on focal cerebral ischemia reperfusion (I/R) injury.
Methods: male Kunming mice were randomly divided into 4 groups: the sham operation group, the I/R model group and the DMY high, low dose group. The model group and the DMY group were used to establish the left middle cerebral artery occlusion (middle cerebral artery occlusion, MCAO) 3h/ reperfusion 24h focal cerebral ischemia reperfusion injury model, and the sham operation group only ligated the left cervical total movement. The.DMY was high in the vein, without the insertion of the thread embolus, and the low dose group continued to intrigastrate DMY500250mg kg-1 before the model, and before the ischemia, 1H, 1H and reperfusion before reperfusion, 6h, 12h respectively 1 times; the ischemia reperfusion group and the sham operation group had the same volume of 0.5% carboxymethyl cellulose in the same volume. After reperfusion 24, the evaluation of the symptoms of neural function defect in mice was evaluated. TTC staining was used to determine cerebral infarction ratio (%), dry wet weight method was used to determine brain water content (%), HE staining was used to observe the pathological changes of brain tissue and count the number of neurons in the cortical semi dark zone. Results: compared with the model group, the DMY250mg kg-1500mg kg-1 group could reduce the death ratio of cerebral infarction after cerebral ischemia injury, and the DMY500mg kg-1 group could also reduce the neurological function deficiency. Loss of score and brain water content, improve the pathological morphology of brain tissue and increase the number of neurons in the cortical semi dark zone (P 0.05 or P 0.01). Conclusion: DMY has a certain protective effect on focal cerebral ischemia reperfusion injury in mice.
The third part of the protective mechanism of two hydrogen myricetin on focal cerebral ischemia-reperfusion in mice
Objective: To study the antioxidant mechanism of two hydrogen myricetin (DMY) on focal cerebral ischemia-reperfusion injury.
Methods: 40 male Kunming mice were randomly divided into 4 groups: the sham operation group, the I/R model group and the DMY high, low dose group, 10.MCAO models in each group and the second part of the DMY administration method. After the ischemia 3H reperfusion 24h, the MDA content and SOD and GSH-PX activity in the ischemic side of the mice were measured respectively.
Results: compared with the model group, DMY500mg kg-1 group could significantly reduce the level of MDA (P 0.01) after cerebral ischemia injury and increase the level of t-SOD and GSH-PX in the brain tissue after I/R injury (P 0.01).
Conclusion: DMY protects mice from focal brain I/R damage and its antioxidant effect.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.31

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