高血糖局灶型脑缺血再灌注损伤磷酸化P38MAPK各亚型的差异性表达

发布时间：2018-04-10 06:37

本文选题：高血糖　切入点：脑缺血再灌注　出处：《宁夏医科大学》2014年硕士论文

【摘要】：目的研究高血糖SD大鼠局灶性脑缺血再灌注后P38MAPKα和β在脑组织中的磷酸化状态，以及在各细胞中的表达和时空规律，，以此探讨P38MAPK在高血糖加重脑缺血损伤中的作用。方法通过腹腔注射STZ制备1型糖尿病模型，线栓法制备大脑中动脉局灶性脑缺血30min/再灌注模型。然后通过HE染色，对比观察糖尿病高血糖局灶脑缺血/再灌注组(高血糖组)和正常血糖局灶脑缺血/再灌注组(正常血糖组)脑组织的病理学变化，神经症状评分了解局灶脑缺血/再灌注后大鼠神经功能缺损情况，采用免疫组化法、免疫荧光染色法和Western Blotting法检测磷酸化P38MAPKα和β(p-p38α，p-p38β)的表达。结果(1)大鼠局灶脑缺血/再灌注术后，大鼠脑缺血后均出现神经功能缺损，高血糖缺血再灌注组的脑功能评分明显较正常血糖手术组升高(P 0.05).(2)光镜下见正常血糖手术组在缺血30min分钟再灌注1d时可见明显脑水肿改变，神经元肿胀、固缩和溶解甚至死亡。再灌注3d时缺血中心区明显神经元、星形胶质细胞溶解坏死，出现空泡结构，再灌注7d时可见脑水肿减轻，神经元减少，部分神经元核淡染，在固缩及肿胀的神经元周围可见胶质细胞的围绕，体积较小的神经胶质细胞增多，缺血半暗带消失，梗死区周边胶质细胞增多。再灌注14d时梗死区范围缩小，没有明显的脑水肿，胶质细胞明显增多，聚集成群，形成胶质细胞结节.高血糖手术组在缺血30min再灌注1d和3d是较正常血糖手术组脑水肿加重，梗死范围扩大，神经元的肿胀、固缩和溶解增加，死亡神经元明显多于正常血糖组，围绕死亡神经元的星形胶质细胞也明显增加。再灌注7d后缺血中心区胶质细胞数量较正常血糖组增加。(3)磷酸化P38MAPK α的免疫组化及Western Blot结果:假手术组及缺血组对侧脑组织仅见p-P38MAPK α的少量表达，缺血组p-P38MAPK α阳性表达均显著高于假手术组及缺血组对侧(P 0.05)。在缺血中心区及周边区，p-P38MAPK α正常血糖组再灌注3d时开始增加，再灌注14d时p-P38MAPK α阳性表达明显增高，达到高峰，至再灌注28d时p-P38MAPK α阳性表达下降。高血糖组p-P38MAPK α在各亚组间阳性表达趋势同正常血糖各亚组间，同时间点高血糖组p-P38MAPK α阳性表达高于正常血糖组(P0.05)。(4)磷酸化P38MAPKβ免疫组化及Western Blot结果：假手术组及缺血组对侧脑组织仅见p-P38MAPK β的少量表达，缺血组p-P38MAPK β阳性表达均显著高于假手术组及缺血组对侧(P 0.05)。在缺血中心区及周边区，缺血组再灌注1d，3d，7d，14d，28d各亚组间p-P38MAPKβ阳性表达无明显差异，同时间点高血糖组p-P38MAPK α阳性表达高于正常血糖组（P0.05）。（5）免疫荧光双标记检测磷酸化p38α和β的结果：早期p-p38α与神经元共表达，后期则与小胶质细胞共表达；p-p38β与神经元共表达。结论（1）在大鼠糖尿病局灶型脑缺血再灌注模型中，高血糖可加重缺血再灌注脑组织损伤，增加脑水肿和脑梗死的面积，加重缺血再灌注引起的神经元变性和凋亡，从而加重大鼠神经系统的症状和体征。（2）在大鼠局灶型脑缺血再灌注模型中，P38MAPKα和β均被激活参与损伤，而高血糖可加重P38MAPKα和β激活程度，从而加重脑缺血再灌注损伤；（3）P38MAPKα和β均可通过加重神经元损害从而加重脑缺血再灌注的损伤；（4）p38MAPK信号通路通过可发生不同程度的磷酸化和激活状态，并通过调节其上下游因子，参与介导高血糖加重脑缺血再灌注损伤。
[Abstract]:Objective to study the phosphorylation state of P38MAPK alpha and beta in brain tissue and the expression of SD and its spatio-temporal patterns in various tissues after focal cerebral ischemia-reperfusion in hyperglycemic rats, so as to explore the role of P38MAPK in hyperglycemia aggravating cerebral ischemia injury.
Methods type 1 diabetes model by intraperitoneal injection of STZ was prepared by the middle cerebral artery, focal cerebral ischemia reperfusion model of 30min/ suture method. Then HE staining, observation of diabetic hyperglycemia in focal cerebral ischemia / reperfusion group (high glucose group) and normal blood glucose in focal cerebral ischemia / reperfusion group (the normal blood glucose group) pathological changes of brain tissue and neurological symptom score to focal cerebral ischemia / reperfusion in rats after nerve function defect, by immunohistochemistry, immunofluorescence staining and Western Blotting method to detect the phosphorylation of P38MAPK alpha and beta (p-p38 alpha, p-p38 beta) expression.
Results (1) in rats with focal cerebral ischemia / reperfusion after cerebral ischemia in rats showed neurological deficit, high blood glucose in ischemia reperfusion group the neurological score was significantly higher than that of normal blood glucose group increased (P 0.05). (2) under the light microscope, the normal blood glucose group in 30min minutes ischemia surgery 1D reperfusion showed obvious changes in cerebral edema, neuron swelling, shrinkage and dissolution and even death. 3D reperfusion in ischemic central area obviously neurons, glial cells astrocytes dissolved necrosis, vacuolar structure, 7d reperfusion visible brain edema, neuron loss, neuronal nuclear staining, visible around the glial cells around pyknosis and swelling of neurons, glial cells with small volume increased, the ischemic penumbra surrounding the infarct area disappeared, glial cells increased. 14d reperfusion infarction area reduced, no obvious brain edema, glial cells increased significantly, poly Integrated group, formation of glial nodules. High blood glucose group in ischemia 30min reperfusion 1D and 3D is lower than normal blood glucose group cerebral edema and infarct expansion, neuron swelling, shrinkage and dissolution increased, neuronal death is significantly higher than the normal blood glucose group, around the dead neurons, astrocytes also increased significantly. The number of 7D after reperfusion in ischemic central area of glial cells than normal glucose group increased (3). Immunohistochemistry and Western Blot results in phosphorylation of P38MAPK alpha: sham operation group and ischemia group on the expression of a small amount of brain tissue only p-P38MAPK alpha, alpha p-P38MAPK expression in ischemia group were significantly higher than those in sham operation group and ischemia group the side (P 0.05). In the ischemic central area and the surrounding area, began to increase p-P38MAPK alpha in normal blood glucose group at 3D after reperfusion, 14d reperfusion p-P38MAPK alpha positive expression was significantly increased, and reached the peak at 28d after reperfusion, to p-P The expression of 38MAPK decreased. High blood glucose group p-P38MAPK alpha in each sub group positive expression trend with normal blood glucose among groups at the same time, high blood glucose group p-P38MAPK positive expression was higher than that of the normal blood glucose group (P0.05). (4) the phosphorylation of P38MAPK beta immunohistochemistry and Western Blot results: sham operation group and the expression of a small amount of ischemia side brain tissue ischemia group only p-P38MAPK beta, p-P38MAPK beta expression was significantly higher than that in sham operation group and ischemia group (P 0.05) on the side. In the ischemic central area and the surrounding area, ischemia reperfusion group 1D, 3D, 7d, 14d, 28d showed no significant difference between the subgroups of p-P38MAPK beta positive expression, at the same time, high blood glucose group p-P38MAPK expression was higher than that of the normal blood glucose group (P0.05). (5) double immunofluorescence detection of phosphorylated p38 alpha and beta alpha and p-p38 results: early neurons co expression, the latter with microglia co expression of p-p38 beta with God; It is expressed in common.
Conclusion (1) in diabetic rat regional cerebral ischemia reperfusion model, hyperglycemia can aggravate the ischemia reperfusion injury of brain tissue, increase cerebral edema and cerebral infarction area, neuronal degeneration and apoptosis increase induced by ischemia reperfusion, which aggravates rat nervous system symptoms and signs in (2). Rat focal cerebral ischemia reperfusion model, P38MAPK alpha and beta are involved in activation and injury, hyperglycemia can aggravate P38MAPK alpha and beta activation, thereby increasing the cerebral ischemia reperfusion injury; (3) P38MAPK alpha and beta can aggravate neuronal damage aggravated from cerebral ischemia reperfusion injury; (4) the degree of phosphorylation and activation occurs via the p38MAPK signaling pathway, and by regulating its downstream factors involved in mediating hyperglycemia and cerebral ischemia reperfusion injury.

【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3

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