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1、三峡大学 硕士学位论文 依达拉奉预处理对大鼠脑缺血再灌注损伤的抗细胞凋亡保护作 用 姓名：赵伟 申请学位级别：硕士 专业：外科学 指导教师：赵东刚 20100501 II 内 容 摘 要 目的：观察大鼠缺血再灌注后大鼠神经功能缺损，梗死灶面积百分比，梗死区神 经元的凋亡指数,与细胞凋亡基因有关的 bcl-2、bax、hsp-70 蛋白表达等变化，运用 依达拉奉预处理进行干预，观察上述指标在大鼠缺血再灌注模型中的表达情况，探讨 依达拉奉预处理对脑缺血再灌注损伤的抗细胞凋亡等神经保护机制， 并为脑血管性疾 病二级预防提供新的途径。 方法：将 SD 大鼠随机分为假手术组，依达拉奉预处理组（预处理组）
2、 ，依达拉奉 后处理组（后处理组） ，预处理对照组，后处理对照组等 5 组。采用改良的 longa 线 栓法建立局灶缺血再灌注模型。造模前 3 天，预处理组预防给予依达拉奉干预，每日 一次，3mg/kg, 采用腹腔注射的方式。造模成功后，术后立即、12h、24h、36h、48h 预处理组、后处理组给予依达拉奉干预。 在采集标本前，对大鼠进行神经功能缺损评分。取标本后，根据 TTC 染色判断梗 死灶面积百分比；HE 染色检测脑组织病理形态学改变；通过 TUNEL 法来检测梗死区 神经元的凋亡指数（AI） ；免疫组化法检测脑组织中 hsp-70 和 Bcl-2、bax 蛋白表达 情况。 结果：脑缺
3、血再灌注大鼠神经功能缺损评分显示，预处理组和后处理组评分与假 手术组比较明显增高，与对照组比较明显降低，而预处理组评分低于后处理组。 TTC 染色结果显示，假手术组无明显梗死现象，预处理组和后处理组较对照组大 脑中动脉供血区梗死面积明显缩小，预处理组梗死面积较后处理组小。 脑组织病理形态学检测表明，光镜下假手术组大鼠皮层各层细胞排列整齐，神经 元结构完整，神经基质无水肿，神经纤维无损坏。对照组大鼠显示大脑皮层细胞排列 紊乱，神经细胞水肿，胞核淡染，核固缩，尼氏小体减少或者消失，树突轴突减少， 血管周围间隙增宽等变化； 预处理组和后处理组见大脑皮质椎体细胞和脑实质神经细 胞结构清楚， 核固缩、
4、核溶解程度较对照组明显减轻， 轻度水肿， 病变范围明显缩小。 TUNEL 法检测结果显示假手术组大鼠大脑皮质未见 TUNEL 阳性细胞。预处理组 和后处理组， 脑缺血再灌注周围皮质可见明显阳性细胞， 与对照组比较有显著差异性。 预处理组在 TUNEL 阳性细胞数少于后处理组。 免疫组织化学技术检测结果显示假手术组大鼠大脑皮质未见 bcl-2、 bax、 hsp-70 阳性细胞；与对照组比较，预处理组和后处理组 bcl-2、hsp-70 蛋白的表达量明显增 多；而 bax 蛋白表达明显减少。预处理组与后处理组比较，bcl-2、bax、hsp-70 蛋 白的表达均有差异性。 结论：在大鼠缺血再灌注
5、模型中，大鼠出现神经功能缺损、梗死灶等表现，给予 III 依达拉奉干预后，大鼠神经功能缺损有所改善、梗死灶缩小等，说明依达拉奉起到了 脑神经保护作用。进一步证实了脑缺血再灌注后, 脑缺血再灌注周围皮质 Bcl-2、 bax、hsp-70 蛋白表达发生了变化，说明细胞凋亡机制参与了脑缺血再灌注损伤。给 予依达拉奉预处理组干预后，Bcl-2、hsp-70 蛋白的表达增加，而 TUNEL 阳性细胞数 量、 bax 蛋白的表达减少； 与后处理比较也有显著差异， 说明依达拉奉能有效抑制 bax 的表达，上调 Bcl-2、hsp-70 的表达，抑制细胞凋亡，减轻脑组织损伤,增强细胞对 缺血再灌注的耐受性。
6、 关键词：凋亡 缺血再灌注 依达拉奉 BCL-2 关键词：凋亡 缺血再灌注 依达拉奉 BCL-2 BAX HSP-70BAX HSP-70 IV Abstract Objective Observed in rats after ischemia-reperfusion in rats neurological deficit, the percentage of infarct size, infarct neuronal apoptosis index, and apoptosis-related genes bcl-2, bax, hsp-70 protein expression an
7、d other changes. By Edaravone pretreatment intervention，Observe the expression of the above indicators in the rat model of ischemia-reperfusion, to probe anti-apoptotic protection mechanisms Of edaravone pretreatment on cerebral ischemia-reperfusion injury, and to provide a new way for the secondary
8、 prevention of cerebrovascular disease. Methods The SD rats were randomly divided into 5 groups： the sham operation group, the edaravone pretreatment group (pretreatment group), the edaravone post-treatment group (post-treatment group), the pretreatment control group, the post-treatment control grou
9、p。The focal middle cerebral artery occlusion (MCAO) model was made by thread-tie method.3d before MCAO, edaravone pretreatment group were given 3mg/kg, once a day, by intraperitoneal injection. The edaravone pretreatment group and After the success of modeling, the edaravone post-treatment group wer
10、e given 3mg/kg, by intraperitoneal injection, at once and at the time of 12h, 24h, 36h, 48h, by intraperitoneal injection. The control group was given normal saline. Before taking samples, The neurological deficits were evaluated in a posture test after the ischemia. After taking samples, according
11、to TTC staining to determine the percentage of infarct size; HE staining was to observate brain tissue pathological changes; by TUNEL method to detect infarct neuronal apoptosis index (AI); by immune staining of brain tissue to Observation expression of hsp - 70 and Bcl-2, bax protein. Results Cereb
12、ral ischemia-reperfusion in rat nerve function deficit score showed that the pretreatment group and the post-treatment group compared with sham group score significantly higher compared with the control group decreased significantly, while the pretreatment group score lower than the post-treatment g
13、roup. TTC staining showed no obvious infarction in sham group phenomenon. The infarction volumes of the pretreatment group and the post-treatment group significantly reduced infarct size compared with the control group. The infarction volumes of the pretreatment group were less than the post-treatme
14、nt group. Brain tissue pathological analysis showed that, in the light microscope, the cell of cortical layers of the sham-operated rats, arranged in neat rows, the structer of neurons was V integrity. There was no edema in neural substrate. Nerve fibers were without damage. The arrangement of corti
15、cal cells of Control rats was disordered. There were edema in nerve cells,and nuclei was pale staining, and nuclear was pyknosis. Nissl body reduced or disappeared, and dendrites axons reduced. The perivascular space widened;, see The cell structure of vertebral cerebral cortex nerve cells and brain
16、 parenchyma of the pretreatment group and the post-treatment group were clear, and nuclear pyknosis and nuclear fusion significantly reduced than the control group. Edema was mild and extent of the lesion significantly reduced compared with the control group. TUNEL assay results showed that sham-operated rats had no TUNEL-positive cells in the cerebral cortex. In the pretreatment group and the post-treatment group, cerebral of ischemia and reperfusion could be seen around the cortex, significa
