Trim27在帕金森病中的表达及其在神经元凋亡中的作用机制研究

发布时间：2018-03-26 13:33

本文选题：帕金森病　切入点：Trim27　出处：《吉林大学》2015年博士论文

【摘要】：目的：细胞凋亡被认为是帕金森病病理机制中多巴胺能神经元的主要死亡方式，但是其中具体的分子机制和信号通路还有待研究。Trim27是Trim家族成员，有显著的促凋亡作用。本文旨在对Trim27在帕金森病中的功能及其可能机制进行探讨，以期为临床帕金森病的早期诊断及有效治疗提供新的有效靶位点。方法：采用实时荧光定量PCR（qPCR）方法对帕金森病患者及正常人外周血中Trim27的表达进行检测；采用qPCR和免疫蛋白印迹（western blot）方法检测Trim27在1-甲基-4-苯基吡啶离子（1-methyl-4-phenylpyridinium，MPP+）诱导的大鼠嗜铬细胞瘤细胞（PC12）和1-甲基-4-苯基-1,2,3,6-四氢吡啶（1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine，MPTP）诱导的帕金森病小鼠黑质致密区的表达情况；采用基因敲除技术结合western blot方法分析探讨Trim27敲除对PC12细胞和帕金森病小鼠神经元凋亡的影响。结果：帕金森病患者外周血中Trim27表达显著高于正常人。50μM MPP+刺激24h能诱导PC12细胞凋亡，细胞凋亡的标志物活化型Caspase-3的表达上升，酪氨酸活化酶TH表达显著下降，，与此同时Trim27表达升高。在PC12细胞中转染Trim27siRNA后，Trim27mRNA和蛋白的表达被有效抑制；MPP+诱导的细胞凋亡明显减少，活化型Caspase-3表达降低。机制研究表明Trim27通过激活NF-κB信号通路来促进细胞凋亡的发生。MPTP成功诱导的帕金森病小鼠模型中，黑质TH阳性多巴胺能神经元显著减少，western blot检测显示诱导后活化型Caspase-3的表达显著上升，且伴随着Trim27表达升高；Trim27-/-小鼠MPTP诱导后，神经元损失减少，凋亡蛋白Caspase-3表达降低。结论： Trim27在帕金森病患者、凋亡的PC12细胞和帕金森病模型小鼠中表达均上升。Trim27的基因沉默使MPP+诱导的PC12细胞凋亡受到抑制，Trim27基因敲除小鼠黑质多巴胺能神经元数量显著高于野生型小鼠，与未诱导组无显著差异，说明，Trim27在帕金森病发生发展过程中发挥了重要作用；Trim27基因敲除显著改善了小鼠帕金森病的状况，提示Trim27可能成为帕金森病治疗的有效新靶点。
[Abstract]:Objective:. Apoptosis is thought to be the main death mode of dopaminergic neurons in the pathophysiology of Parkinson's disease, but the specific molecular mechanisms and signaling pathways remain to be studied. Trim27 is a member of the Trim family. The purpose of this study is to investigate the function and possible mechanism of Trim27 in Parkinson's disease, in order to provide a new effective target for early diagnosis and effective treatment of Parkinson's disease. Methods:. The expression of Trim27 in peripheral blood of patients with Parkinson's disease and normal subjects was detected by real-time fluorescence quantitative PCR. The expression of Trim27 in the substantia nigra compact region induced by 1-methyl-4-phenylpyridinium-MPP in rat pheochromocytoma cells (PC12) and 1-methyl-4-methyl-4-phenyl-1-methyl-4-phenyl-1-tetrahydropyridine (MPP) induced by 1-methyl-4-phenylpyridine (1-methyl-4-phenylpyridyl) and 1-methyl-4-phenyl-6-tetrahydropyridine (MPP) in the substantia nigra of Parkinson's disease mice was detected by qPCR and Western blot. The effects of Trim27 knockout on apoptosis of PC12 cells and neurons in Parkinson's disease mice were studied by gene knockout technique and western blot method. Results:. The expression of Trim27 in peripheral blood of patients with Parkinson's disease was significantly higher than that of normal controls (.50 渭 M MPP) for 24 hours. The expression of activated Caspase-3, a marker of apoptosis, was increased, and the expression of tyrosine activase th was significantly decreased. At the same time, the expression of Trim27 was increased. After transfection of Trim27siRNA in PC12 cells, the expression of trim27 mRNA and protein was effectively inhibited. The expression of activated Caspase-3 was decreased. The mechanism of Trim27 promoting apoptosis by activating NF- 魏 B signaling pathway. MPTP successfully induced Parkinson's disease mice model. Substantia nigra th positive dopaminergic neurons significantly decreased the expression of activated Caspase-3 after induction by western blot. With the increase of Trim27 expression, the loss of neurons and the expression of apoptotic protein Caspase-3 decreased after MPTP induction. Conclusion:. Trim27 in Parkinson's disease, The gene silencing of apoptotic PC12 cells and Parkinson's disease model mice increased the number of dopaminergic neurons in substantia nigra of PC12 cells induced by MPP, and the number of dopaminergic neurons in substantia nigra of MPP knockout mice was significantly higher than that in wild-type mice. There was no significant difference between the two groups, which suggested that Trim27 gene knockout played an important role in the pathogenesis and development of Parkinson's disease. The knockout of Trim27 gene significantly improved the status of Parkinson's disease in mice, suggesting that Trim27 might be an effective new target for the treatment of Parkinson's disease.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R742.5

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