低血糖性脑损伤蛋白组学分析及DJ-1蛋白功能研究

发布时间：2018-02-24 20:29

  本文关键词： 低血糖 脑损伤 iTRAQ 自噬 DJ-1　出处：《上海交通大学》2015年博士论文　论文类型：学位论文

【摘要】：背景低血糖是糖尿病患者强化血糖控制的常见并发症。低血糖最容易导致脑损伤。“葡萄糖再灌注性脑损伤”概念的提出,使得人们对低血糖性脑损伤发病机制的认识更加复杂,也引发了对传统临床低血糖救治的再思考。研究低血糖发作及恢复过程中与脑损伤有关的病理机制,有可能为临床低血糖救治过程中提供脑保护治疗的靶标和思路。目的本研究旨在采用同位素相对标记与绝对定量技术(Isobaric tag for relative and absolute quantitation,ITRAQ)连同多维液相色谱-串联质谱联用技术(LC-MS/MS)寻找低血糖大鼠与正常大鼠脑组织内的差异表达蛋白,对其进行表达量的验证及相关生物学功能探讨,初步阐明低血糖性脑损伤发病的相关分子机制,为低血糖性脑损伤的治疗提供理论依据。方法注射胰岛素建立低血糖大鼠脑损伤模型,提取脑组织蛋白,酶解后用i TRAQ化学标签标记,进行多维液相色谱分离—串联质谱鉴定,筛选潜在的可能与低血糖性脑损伤相关的差异蛋白。选取差异表达明显且生物学功能相关者在重新建立的动物模型上进行m RNA和蛋白水平的表达量验证。最后在模拟低血糖的体外无糖培养星形胶质细胞模型上利用RNA干扰技术进行相关生物学功能的初步研究。结果通过比较低血糖大鼠与假造模大鼠脑组织的蛋白表达,以相对表达量≥1.5或≤0.67为标准,共筛选到147种差异表达蛋白。采用实时荧光定量聚合酶链反应和免疫印迹法在复制的动物模型上证实AQP4,NDRG1,DJ-1和Clusterin四个蛋白的表达量与在蛋白组学中发现的表达量变化趋势一致。在模拟的体外星形胶质细胞缺糖模型上进一步验证了缺糖上调AQP4和DJ-1两个蛋白的表达量。沉默DJ-1蛋白后,星形胶质细胞在无糖培养时死亡率明显增加。将星形胶质细胞进行无糖培养,观察到AMPK通路被激活,自噬被启动,但自噬通量受损。工具药抑制自噬后无糖培养致星形胶质细胞死亡明显增加。与正常表达DJ-1的细胞比较,沉默DJ-1表达的星形胶质细胞在无糖培养时AMPK通路受损,m TOR活性升高,自噬活性明显降低。结论DJ-1蛋白在低血糖性脑损伤中发挥保护作用,减少缺糖导致的星形胶质细胞损伤,其部分机制通过调节自噬来实现。
[Abstract]:Background hypoglycemia is a common complication of intensive blood glucose control in diabetic patients. Hypoglycemia is most likely to lead to brain injury. The concept of "glucose-reperfusion brain injury" has made people's understanding of the pathogenesis of hypoglycemic brain injury more complicated. It also triggered a reconsideration of the traditional clinical treatment of hypoglycemia. The pathological mechanism related to brain injury during hypoglycemia attack and recovery was studied. Objective the aim of this study was to use isotopic tag for relative and absolute quantitative technique to study Isobaric tag for relative and absolute quantification of Isobaric and Isobaric for relative and absolute, and multidimensional liquid chromatography-tandem substance. [WT5 "HZ] [WT5" HZ] [WT5 "BZ] [WT5" HZ]. LC-MS / MS was used to search for differentially expressed proteins in the brain of hypoglycemic rats and normal rats. The molecular mechanism of hypoglycemic brain injury was preliminarily elucidated by the verification of its expression and the discussion of its related biological functions. Methods the model of hypoglycemic brain injury in rats was established by insulin injection. Brain tissue proteins were extracted and labeled with I TRAQ chemical label after enzymatic hydrolysis, and identified by multi-dimensional liquid chromatography-tandem mass spectrometry. Potential differentially expressed proteins associated with hypoglycemic brain injury were screened. The expression levels of m RNA and protein were tested on the reestablished animal model with distinct differential expression and biological functional correlation. RNA interference technique was used to study the biological function of hypoglycemic astrocytes in vitro. Results the expression of protein in the brain of hypoglycemic rats was compared with that of hypoglycemic rats. If the relative expression is 鈮，

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