人工合成E-选择素对大鼠局灶性脑缺血再灌注损伤血脑屏障的影响及机制探讨

发布时间：2018-04-03 03:12

本文选题：线栓法　切入点：脑缺血再灌注损伤　出处：《苏州大学》2014年硕士论文

【摘要】：目的：使用改良Zea Longa线栓法建立SD (Sprague-Dawley)局灶性脑缺血再灌注损伤大鼠模型，应用人工合成E-选择素对大鼠进行干预，研究其对大鼠脑缺血再灌注损伤后的血脑屏障通透性、脑梗死及脑水肿体积的影响。方法：将90只雄性SD大鼠随机分为假手术组（A组）、模型组（B组）和人工合成E-选择素干预组（C组），每组30只。采用改良Zea Longa线栓法建立SD大鼠脑缺血再灌注损伤模型。E-选择素干预组在建立模型10min前从股静脉注射E-选择素（10mg/kg）。所有大鼠在取脑前2h从股静脉注射2.5%伊文氏蓝（0.2mg/kg）。在缺血2h再灌注3h、6h、24h、48h、72h5个时间点进行神经行为学评分、MRI扫描测定脑梗死及脑水肿的体积比、多功能酶标仪测定脑组织中伊文氏蓝含量等方法了解人工合成E-选择素对脑缺血再灌注损伤大鼠血脑屏障通透性的干预作用。结果：（1） A组未出现神经功能缺失，B组和C组均有不同程度的神经功能缺失，B组与C组之间部分时间点（3h、6h、24h）神经功能评分存在统计学差异（P0.05）；（2）A组无脑水肿及梗死， B、C两组均有不同程度脑水肿及梗死且C组各时间点MRI测得脑组织梗死及水肿的体积较B组小，有统计学意义（P0.05）；（3）A组脑组织伊文氏蓝含量各时间点无明显变化，B组和C组两组变化趋势相近，再灌注3h EB含量开始升高，6h继续升高，24h达到峰值，48h有所下降，，72h仍高于A组。C组各时间点伊文氏蓝含量值均低于B组，两组差异显著（P0.05）。结论：1、大鼠局灶性脑缺血再灌注损伤可导致血脑屏障的通透性增加；2、人工合成E-选择素能够减少大鼠局灶性脑缺血再灌注损伤导致的脑梗死及脑水肿的体积，减轻血脑屏障破坏导致的通透性增加，具有血脑屏障保护作用。目的：探讨人工合成E-选择素对大鼠局部脑缺血再灌注损伤脑组织内皮细胞间紧密连接蛋白咬合蛋白（occludin）及闭锁小带蛋白-1（ZO-1）含量表达及位置结构的影响，从而初步探讨人工合成E-选择素对血脑屏障保护的作用机制。方法：采用改良的Zea Longa线栓法建立SD大鼠局灶性脑缺血再灌注损伤模型，将90只雄性SD大鼠随机分为假手术组（A组）、模型组（B组）和人工合成E-选择素干预组（C组），每组30只。各组大鼠分别在脑缺血2h再灌注3h、6h、24h、48h、72h5个时间点提取脑组织标本。采用免疫组化法观察缺血区脑组织occludin及ZO-1的位置结构及含量，Western blot法半定量测定occludin及ZO-1的含量。结果：免疫组化：（1）A组occludin及ZO-1阳性细胞均沿血管丰富表达。（2）B组occludin及ZO-1都于缺血再灌注3h阳性表达数减少，24h阳性表达数降至最低，72h阳性表达有所增加，但仍低于A组。occludin表达的连续性在24h达到最差。（3） C组occludin及ZO-1阳性表达数在各时间点均高于B组但低于A组。Occludin表达的连续性在24h也好于B组，差于A组。 Western blot半定量分析结果：（1）A组occludin及ZO-1蛋白表达水平无明显变化。（2）B组occludin及ZO-1蛋白的变化趋势相似，3h蛋白表达水平开始减少，6h继续减少，24h达到最低值，48h有所回升，72h仍低于A组。（3）C组各时间点occludin及ZO-1变化趋势同B组，但蛋白表达水平均高于B组，差异均有统计学意义（P0.05）。结论：1．局灶性脑缺血再灌注损伤后大鼠脑组织内皮细胞紧密连接蛋白occludin及ZO-1表达减少；2．人工合成E-选择素血脑屏障的保护作用可能与其抑制occludin及ZO-1表达的减少以及维持occludin位置和结构有关。
[Abstract]:Objective: to establish a method using modified Zea SD plug Longa line (Sprague-Dawley) rat model of focal cerebral ischemia reperfusion injury, the application of artificial synthetic E- selectin intervention in rats, study on rat cerebral ischemia reperfusion blood brain barrier permeability after injury and effect of cerebral infarction and cerebral edema volume.
Methods: 90 male SD rats were randomly divided into sham operation group (A group), model group (group B) and synthetic E- selectin in the intervention group (C group), 30 rats in each group. The establishment of cerebral ischemia reperfusion damage in SD rat model of.E- selectin in intervention group from femoral vein injection of E- in model 10min the former using modified Zea Longa suture method (10mg/kg). All rats in the brain before 2H was injected into the femoral vein, 2.5% Evans blue (0.2mg/kg). After 2h of ischemia reperfusion 3h, 6h, 24h, 48h, 72h5 time point neurobehavioral score, cerebral infarction and cerebral determination edema MRI scan volume ratio method of Evans blue content of multifunctional microplate in brain tissue were determined to investigate the intervention effect of synthetic E- selectin on cerebral ischemia reperfusion injury of blood brain barrier permeability in rats.
Results: (1) A group without loss of nerve function, neurological deficits in different degrees of B group and C group, B group and C group between the time point (3H, 6h, 24h) there was a significant difference between the scores of neural function (P0.05); (2) A group without cerebral edema and infarction, B C, the two groups have different levels of cerebral edema and infarction and C group at each time point MRI measured the brain infarct and edema volume was smaller than that in group B, with statistical significance (P0.05); (3) the brain tissue of the A group of Evans blue content at each time point has no obvious change, B group and C group of two change similar trends, reperfusion 3H EB content began to increase, 6h continues to rise, 24h peak, 48h decreased, 72h is still higher than that of A group.C group at different time points of Evans blue content were lower than B group, there was significant difference between two groups (P0.05).
Conclusion: 1, can lead to increased permeability of the blood-brain barrier in rats with focal cerebral ischemia reperfusion injury; 2, synthetic E- selectin can reduce rat focal cerebral ischemia reperfusion injury caused by cerebral infarction and cerebral edema volume, reduce the destruction of the blood-brain barrier permeability leading to increased with protection the role of the blood-brain barrier.
Objective: To investigate the effect of synthetic E- selectin on local cerebral ischemia reperfusion injury in rat brain endothelial cell tight junction protein occludin (occludin) and -1 (ZO-1) zonula occludens protein content expression and location of structure, so as to investigate the synthetic E- mechanism of selectin on protection of blood brain barrier.
Methods: SD rats with focal cerebral ischemia reperfusion injury model using Zea Longa modified suture method, 90 male SD rats were randomly divided into sham operation group (A group), model group (group B) and synthetic E- selectin in the intervention group (C group), 30 rats in each group. All the rats were 2H in cerebral ischemia reperfusion 3h, 6h, 24h, 48h, 72h5 time points to extract brain tissue specimens. The position of structure and content by immunohistochemical staining of occludin and ZO-1 in brain tissue was observed in ischemic area, semi quantitative determination of occludin content and ZO-1 Western blot method.
Results: the results of immunohistochemistry: (1) A group occludin and ZO-1 positive cells were abundant along vascular expression. (2) B and ZO-1 in group occludin ischemia reperfusion 3H positive expression decreased, 24h expression decreased to the lowest number, the positive expression of 72h increased, but still lower than the continuous expression of A group.Occludin lowest in 24h. (3) C group occludin and ZO-1 positive expression at each time point were higher than that of B group but lower than the continuous expression of A.Occludin in 24h group or in B group, the difference in the A group.
Western blot semi quantitative analysis results: (1) A group occludin and the expression level of ZO-1 protein had no obvious change. (2) the change trends of the B group of occludin and ZO-1 protein, 3h protein expression level began to decrease, 6h 24h continued to decline, reaching the lowest value of 48h increased, 72h is still lower than that of A group (3. C) group at different time points occludin and ZO-1 changes with the B group, but the expression level was higher than that of B group, the differences were statistically significant (P0.05).
Conclusion: 1. focal cerebral ischemia reperfusion injury after rat brain endothelial cell tight junction protein occludin and ZO-1 expression decreased; protective effect of 2. synthetic E- selectin blood brain barrier may be reduced due to the inhibition of occludin and expression of ZO-1 and occludin to maintain position and structure.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3

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