自噬相关基因Beclin-1、ATG7、ATG3、LC3在脑缺血再灌注大鼠脑组织的表达

发布时间：2018-08-17 20:07

【摘要】：目的探讨局灶性脑缺血再灌注(ischemia reperfusion,IR)损伤时,受损大脑皮质中bcl-2同源结构域蛋白(Beclin-1)、自噬相关基因7(autophagy related gene7,ATG7)、自噬相关基因3(autophagy related gene 3,ATG3)和自噬微管相关蛋白轻链3(microtubule-associated protein1 light chain3,LC3)的表达规律。方法将健康雄性SD大鼠随机分为假手术组(sham组)和模型组(IR组)。其中模型组又分为再灌注IR 0h、1h、3h、6h、12h、24h六个亚组。采用大脑中动脉栓塞术(middle cerebral artery occlusion,MCAO)制备局灶性脑缺血再灌注损伤模型。动物苏醒后采用神经功能缺损评分,模型制备24 h后采用2,3,5-三苯基氯化四氮唑(2,3,5-Triphenyltetrazolium chloride,TTC)染色及脑组织含水量方法鉴定模型是否成功。采用透射电镜观察各组大鼠大脑神经元的自噬情况,采用免疫组化和免疫印迹方法检测大脑皮质Beclin-1、ATG7、ATG3和LC3的表达规律。结果模型鉴定结果显示:sham组大鼠的行为学表现正常,神经功能未见缺损症状,脑组织未见水肿和梗死区;而IR各组大鼠均出现明显的神经功能缺损症状,IR 12h后脑组织含水量显著增高,IR 6h后可见明显的苍白梗死区,与sham组比较有显著差异。透射电镜sham组可见正常的神经元形态,而IR各组均可见到明显的神经元自噬。免疫组化染色后发现,sham组仅见少量Beclin-1阳性表达细胞,而IR不同时间点则呈现不同的表达增高趋势,其中IR 3h和IR 24h组呈现较明显的表达。免疫印迹检测Beclin-1也呈现一致的表达趋势。免疫组化和免疫印迹结果显示,ATG7、ATG3、LC3表达也呈现与Beclin-1一致的趋势。结论Beclin-1、ATG7、ATG3、LC3在调节脑缺血再灌注损伤中的细胞自噬具有重要的作用。
[Abstract]:Objective to investigate the expression of bcl-2 homologous domain protein (Beclin-1), autophagy associated gene 7 (autophagy related gene7 ATG7, autophagy associated gene 3 (autophagy related gene 3, and microtubule-associated protein1 light chain3LC3 in the damaged cerebral cortex during focal cerebral ischemia-reperfusion (ischemia) injury. Methods healthy male SD rats were randomly divided into sham operation group (sham group) and model group (IR group). Among them, the model group was divided into six subgroups of IR 0 h, 1 h, 3 h, 6 h, 12 h and 24 h. Middle cerebral artery embolization (middle cerebral artery occlusion MCAO) was used to establish focal cerebral ischemia-reperfusion injury model. After recovery, neurological deficit score was used to evaluate the success of the model 24 hours later, and the TTC staining and the water content of brain tissue were used to determine the success of the model. The autophagy of cerebral neurons in each group was observed by transmission electron microscope, and the expression of ATG3 and LC3 in cerebral cortex were detected by immunohistochemistry and immunoblotting. Results the results of model identification showed that the behavior of rats in the control group was normal, the neurological function was not impaired, no edema and infarct area were found in the brain tissue. After 12 hours of IR, the water content of brain tissue was significantly increased. After 6 hours of IR, pallid infarct area could be seen, which was significantly different from that of sham group. The morphology of neurons was normal in sham group, but obvious autophagy was observed in IR group. After immunohistochemical staining, only a small number of Beclin-1 positive cells were found in the control group, but the expression of IR increased at different time points, especially in the IR 3 h and IR 24 h groups. Western blot analysis of Beclin-1 also showed a consistent trend of expression. Immunohistochemistry and Western blotting showed that the expression of ATG7, ATG3 and LC3 was consistent with that of Beclin-1. Conclusion Beclin-1 ATG7 ATG3 + LC3 plays an important role in regulating autophagy in cerebral ischemia-reperfusion injury.
【学位授予单位】：锦州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.3

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