雷帕霉素对大鼠局灶性脑缺血再灌注脑水肿作用及机制的研究

发布时间：2018-05-11 06:52

本文选题：雷帕霉素 + 脑缺血　；参考：《滨州医学院》2014年硕士论文

【摘要】：引文脑缺血再灌注后梗死灶周围的脑组织存在炎症反应及水肿。炎症反应可以导致脑细胞受损,从而导致并加重脑水肿。炎症反应中的某些炎症因子可以影响脑组织中AQP4及MMP9蛋白的表达。AQP4在血脑屏障的水分子转运中起重要作用,MMP9在BBB基底膜破坏中起重要作用。这两种蛋白表达增多时可以加重脑缺血再灌注后脑水肿的程度,严重影响患者的病情变化及预后。目的研究大鼠脑缺血再灌注同时应用雷帕霉素对大鼠脑梗死体积、脑水肿程度、BBB受损程度及病理学变化、AQP4及MMP9蛋白的表达量等的影响,探讨雷帕霉素对大鼠局灶性脑缺血再灌注损伤后的脑水肿是否具有保护作用,并初步分析雷帕霉素发挥保护的机制。方法将72只成年雄性SD大鼠随机分为三组,雷帕霉素(rapamycin, RAPA)组、二甲基亚砜(dimethyl sulfoxide, DMSO)组和假手术(Sham)组,每组24只。在激光多普勒血流仪(laser doppler flowmeter, LDF)实时监测下,对RAPA组、DMSO组大鼠应用线栓法制作大脑中动脉闭塞(MCAO)模型,缺血1h后行再灌注,RAPA组、DMSO组分别于再灌注开始时经腹膜腔注射250 μg/ml的RAPA稀释液(250 μg/kg体重)、DMSO (1 ml/kg体重)；Sham组大鼠只模拟手术,不插入栓线。各组大鼠于麻醉清醒后进行神经功能缺损评分,于再灌注48 h后取材行脑梗死体积(6只)、脑组织含水量(6只)、血脑屏障(blood-brain barrier, BBB)通透性的测定(6只),光镜、电镜对脑组织进行形态学观察,免疫组织化学法检测大脑皮质水通道蛋4 (aquaporin 4, AQP4)和金属基质蛋白酶9 (matrix metalloproteinase 9, MMP9)的表达(6只)。结果①RAPA组脑梗死体积小于DMSO组(P0.05)。 ②RAPA组脑组织含水量低于DMSO组(P0.05)。 ③RAP A组伊文思蓝(Evans blue, EB) BBB的透出量明显低于DMSO组(P0.01)。④MCAO模型组脑组织损伤位于左侧额叶及顶叶大脑皮层。DMSO组梗死区域内可见大量的正常神经元变性、坏死,甚至核固缩、尼氏体消失,形成“红色神经元”(嗜酸性变性)；脑组织水肿明显,大量中性粒细胞浸润,毛细血管周围间隙明显增大。RAPA组梗死范围及缺血半暗带范围明显小于DMSO组,脑组织缺血及梗死等病理变化程度较DMSO组轻。假手术组脑组织结构基本正常。⑤DMSO组梗死区血脑屏障完整性严重受损,毛细血管管腔闭锁,管周水肿明显,基膜欠完整。RAPA组BBB破坏程度较对照组轻。⑥RAPA组大脑半球额、顶叶皮层缺血半暗带区AQP4、MMP9的表达均明显低于DMSO组(P0.01)。结论再灌注同时应用雷帕霉素可以减小脑梗死体积,减轻脑水肿程度,降低BBB的病理学损伤程度,还可以降低脑组织中AQP4和MMP9的表达。雷帕霉素可能是通过抑制免疫级联反应,下调脑组织中AQP4和MMP9的表达,改善脑水肿,从而对缺血再灌注损伤后的脑组织产生保护作用。
[Abstract]:There are inflammatory reaction and edema in the brain tissue around the infarct after cerebral ischemia reperfusion. Inflammation can cause brain cells to be damaged, leading to and exacerbating brain edema. Some inflammatory factors may affect the expression of AQP4 and MMP9 protein in brain tissue. AQP4 plays an important role in the water molecule transport of blood-brain barrier. MMP9 plays an important role in the destruction of BBB basement membrane. The increased expression of these two proteins can aggravate the degree of cerebral edema after cerebral ischemia reperfusion, and seriously affect the state of disease and prognosis of the patients. Objective to study the effects of rapamycin on cerebral infarction volume, cerebral edema and pathological changes in rats with cerebral ischemia-reperfusion and the expression of AQP4 and MMP9 protein. To investigate the protective effect of rapamycin on brain edema after focal cerebral ischemia reperfusion injury in rats, and to analyze the protective mechanism of rapamycin. Methods Seventy-two adult male Sprague-Dawley rats were randomly divided into three groups: rapamycin (rapamycin) group, dimethyl sulfoxide (DMSO) group and sham operation group (24 rats in each group). The model of middle cerebral artery occlusion (MCAO) was established by using the method of thread occlusion in rats of RAPA group under the real-time monitoring of laser doppler flowmeter, LDF) with laser Doppler flow meter. At the beginning of reperfusion, the rats in the rapa group were injected with 250 渭 g/ml RAPA diluted solution (250 渭 g/kg) at the beginning of reperfusion, respectively. The rats in the control group were given DMSO 1 ml/kg weight / body weight respectively. The rats in the sham group were treated with simulated operation and no thrombus was inserted. The rats in each group were assessed for neurological impairment after anaesthesia. After 48 hours of reperfusion, 6 rats with cerebral infarction volume, 6 rats with cerebral tissue water content and 6 rats with blood-brain barrier permeability were measured under light microscope. The expression of aquaporin 4 (AQP4) and metalloproteinase 9 matrix metalloproteinase 9 (MMP9) in cerebral cortical aquaporin 4 (AQP4) and metalloproteinase 9 (MMP9) were detected by immunohistochemistry. Results the volume of cerebral infarction in 1RAPA group was smaller than that in DMSO group (P 0.05). The brain tissue water content in 2RAPA group was lower than that in DMSO group (P 0.05). The permeability of Evans blue, EB) BBB in 3RAP A group was significantly lower than that in DMSO group (P 0.01) .4MCAO model group was located in left frontal lobe and parietal area. In lobar cortex. DMSO group, a large number of normal neuronal degeneration could be seen in the infarcted area. Necrosis, even nuclear shrinkage, the disappearance of the Nissl body, and the formation of "red neurons" (eosinophilic denaturation); brain tissue edema, massive neutrophil infiltration, The infarct size and ischemic penumbra in the rapa group were significantly smaller than those in the DMSO group, and the pathological changes of cerebral ischemia and infarction were lighter in the rapa group than in the DMSO group. In sham-operation group, the integrity of blood-brain barrier in infarcted area was seriously damaged, capillary lumen atresia, peritube edema was obvious, and the degree of BBB damage in rapa group was less than that in control group. The expression of AQP4 MMP9 in ischemic penumbra of parietal cortex was significantly lower than that in DMSO group. Conclusion reperfusion with rapamycin can reduce the volume of cerebral infarction, reduce the degree of cerebral edema, reduce the degree of pathological injury of BBB, and decrease the expression of AQP4 and MMP9 in brain tissue. Rapamycin may decrease the expression of AQP4 and MMP9 in brain tissue by inhibiting immune cascade reaction, and improve brain edema, which may have protective effect on brain tissue after ischemia-reperfusion injury.
【学位授予单位】：滨州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743;R742

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