携带A53T突变人α突触核蛋白转基因帕金森病大鼠模型的建立
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国家“重大新药创制”科技重大专项课题“啮齿类研发平台创新药物研究开发技术平台建设”(2011ZX09307-302); 国家科技支撑计划课题“神经和代谢疾病基因工程模型的建立”(2012BAI39B02)


Establishment of α - synuclein with A53T mutation Transgenic rat Model of Parkinson’s Disease
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    目的 大鼠在神经系统疾病模型方面比小鼠有更好的表现,建立人α–syn A53T突变型转基因大鼠模型,为研究帕金森病发病提供更优良的疾病模型。方法 构建人α–syn A53T表达载体,显微注射法制备转基因大鼠。PCR法鉴定转基因首建鼠及其子代基因型。Western blotting检测转基因大鼠脑组织中人α – syn蛋白的表达。免疫组化和免疫荧光观察中脑黑质酪氨酸羟化酶阳性神经元数目变化,观察人α – syn蛋白在转基因大鼠黑质神经元中的表达和分布。Rotating rod实验和步态分析系统评价转基因大鼠的行为学改变。结果 获得1个α- syn A53T高表达且可以稳定传代的转基因大鼠品系。转基因大鼠中脑黑质酪氨酸羟化酶(TH)阳性神经元数目减少69% (P<0.05),残存神经元胞浆中有大量的α- syn蛋白聚集。转基因大鼠在滚轴上保持平衡的时间显著减少40% - 60% (P<0.05), 并表现出步态障碍如步宽加大、摆动期缩短和足迹最大接触面积减小等。结论 建立了人α – syn A53T突变型转基因的帕金森病大鼠模型。

    Abstract:

    Objective In view of better performance of rat than mice in nervous diseases studies, to generate a transgenic Parkinson's disease rat line expressing alpha-synuclein gene with A53T mutation and provide a better PD animal model. Methods The transgenic plasmid was constructed by inserting the alpha-synuclein gene with A53T mutation into the downstream of mouse PDGF promoter. The transgenic rats were produced by microinjection and the genotype was detected by PCR. The protein expression levels in brain tissues were determined by western blotting. The expression of human alpha -synuclein in the brain tissues from transgenic rats was analyzed by immunohistochemistry staining(IHC) and immunofluorescence(IF).The changes of the number of tyrosine hydroxylase(TH)-positive neurons in the substantia nigra were observed by IHC and IF. The motor performances of transgenic rats were measured by rotating rod experiment and gait analysis system. Results One transgenic rat line with high human alpha-synuclein expression levels was established. Histopathological examination revealed the abnormal aggregation in midbrain dopamine (DA) neurons and significantly reduced number of TH-positive neurons (69%,P<0.05) in substantia nigra from transgenic rats. Rotating rod and rat gait analysis indicated declined motor performances(40% - 60%,P<0.05)) and gait disorder of transgenic rats. Conclusions The human a-synuclein A53T transgenic rat models of Parkinson’s disease were established.

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张丽,陈炜,张旭,孙秀萍,杨亚军,张连峰.携带A53T突变人α突触核蛋白转基因帕金森病大鼠模型的建立[J].中国比较医学杂志,2013,23(10).

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  • 收稿日期:2013-07-26
  • 最后修改日期:2013-08-02
  • 录用日期:2013-09-22
  • 在线发布日期: 2013-12-16
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