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宋娜,王海军,李宏彬,邵明龙,张伟,谷腾腾,胡庆,余建,张景航,苏蔚,赵铁锁,李雨姗.建立人α 突触核蛋白A30P 突变的帕金森病大鼠模型[J].中国实验动物学报,2018,26(5):567~573.
建立人α 突触核蛋白A30P 突变的帕金森病大鼠模型
Establishment of a transgenic rat model of Parkinson’s disease with an α?synuclein A30P mutation
投稿时间:2018-04-04  
DOI:10.3969/j. issn. 1005 - 4847. 2018. 05. 005
中文关键词:  α?突触核蛋白  A30P  慢病毒表达载体  帕金森病  大鼠
英文关键词:α?synuclein  A30P  lentiviral expression vector  Parkinson’s disease  rat
基金项目:
作者单位E-mail
宋娜 新乡医学院基础医学院 sarah_song1986@163. com 
王海军 新乡医学院基础医学院  
李宏彬 新乡医学院公共卫生学院  
邵明龙 河南省精神病医院,河南省生物精神病学重点实验室,新乡医学院第二附属医院  
张伟 新乡医学院基础医学院  
谷腾腾 新乡医学院基础医学院  
胡庆 新乡医学院基础医学院  
余建 新乡医学院第一附属医院
河南新乡 453003 
 
张景航 新乡医学院第一附属医院
河南新乡 453003 
 
苏蔚 新乡医学院第一附属医院
河南新乡 453003 
 
赵铁锁 新乡医学院基础医学院  
李雨姗 新乡医学院公共卫生学院 limanandliman@163. com 
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中文摘要:
      目的 建立人α?突触核蛋白(α?synuclein,α?SYN)A30P 突变型转基因大鼠的帕金森病(Parkinson’s disease, PD)模型?方法 利用慢病毒系统构建过表达野生型α?SYN 载体pLKO?CMV?α?SYN?WT?P2A?GFP 及α?SYN A30P 突变载体pLKO?CMV?α?SYN?A30P?P2A?GFP,分别转染293FT 细胞,瞬时转染24 h 后WB 检测α?SYN 的表达水平?之后经慢病毒包装?浓缩,利用立体定位技术分别对大鼠中脑黑质致密部注射病毒稀释液,过表达α?SYN 野生型和A30P 突变型的慢病毒颗粒?免疫荧光染色检测α?SYN 和酪氨酸羟化酶(tyrosine hydroxylase, TH)的分布情况,并观察中脑黑质致密部多巴胺能神经元数量的变化?Rotating rod 实验评价注射A30P 慢病毒大鼠的行为改变情况?结果 野生型和突变型A30P 的基因表达载体在293FT 细胞内能够高表达α?SYN 蛋白?TH 免疫荧光结果显示:与病毒稀释液组相比,过表达α?SYN 野生型和A30P 突变型大鼠均能导致中脑黑质致密部多巴胺能神经元数量的减少,而α?SYN A30P 慢病毒注射组的中脑黑质神经元缺失的更多,具有显著性差异?对A30P 转基因大鼠脑部神经元缺失部位进行免疫荧光发现,该区域TH 染色几乎呈阴性,α?SYN 蛋白出现大量聚集,该结果表明脑部神经元的消失同时伴随着α?SYN 蛋白的聚集及TH 表达的剧烈减少,进一步揭示过表达α?SYN A30P 可以导致多巴胺能神经元数量的减少和退化?此外,rotating rod 实验结果显示过表达α?SYN A30P 大鼠表现出明显的进行性运动能力障碍?结论 通过慢病毒系统建立了人α?SYN A30P 突变型转基因大鼠的PD 模型,为PD 发病机制的研究及药物开发奠定基础?
英文摘要:
      Objective The aim of this study was to develop a transgenic rat model of Parkinson’s disease by lentiviral vector?mediated overexpression of human α?synuclein in the substantia nigra of rats. Methods Lentiviral expression vectors were constructed by inserting the normal α?synuclein gene (WT) or the α?synuclein gene with an A30P mutation (A30P) downstream of the Cytomegalovirus (CMV) promoter (pLKO?CMV?α?SYN?WT?P2A?GFP and pLKO2?CMV?α?SYN?A30P?P2A?GFP). The vectors were transduced into 293FT cells, and α?synuclein protein levels were detected by western blotting after transient transfection for 24 h. Following the packaging, enrichment, the viral suspension with control, and normal or mutated forms of α?synuclein, were stereotaxically injected into the substantia nigra. After 21 days, the rats were euthanized, and the substantia nigra harvested and processed for immunofluorescence to detect α?synuclein and tyrosine hydroxylase (TH) expression, and changes in numbers of neurons. Motor performance was also assessed in the A30P rats using the rotating rod test. Results Each lentiviral vector induced equivalent levels of normal or A30P mutated α?synuclein expression. Compared with the control group, both the wild type and A30P mutant groups showed an obvious reduction in numbers of dopaminergic neurons in the substantia nigra, with significantly greater injury in the A30P group. Immunofluorescence staining experiments in the injured region of A30P mutant rats showed largely absent TH staining, but abundant α?synuclein immunoreactive aggregation. The human α?synuclein group was also associated with a marked reduction in TH expression, indicating that A30P mutant overexpression caused neuronal degeneration. In addition, A30P mutant rats showed a progressive decline in motor performance. Conclusions We have established a human α?synuclein A30P transgenic rat model of Parkinson’ s disease, which may be useful for understanding the pathogenesis and developing treatments for this disorder.
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