中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2017, Vol. 12 ›› Issue (8): 1347-1351.doi: 10.4103/1673-5374.213557

• 原著:退行性病与再生 • 上一篇    下一篇

Pitx3基因敲低可影响多巴胺能神经细胞中胶质源性神经营养因子的转录活性

  

  • 收稿日期:2017-06-20 出版日期:2017-08-15 发布日期:2017-08-15
  • 基金资助:

    国家自然科学基金(81372698).

Impact of Pitx3 gene knockdown on glial cell line-derived neurotrophic factor transcriptional activity in dopaminergic neurons

 Jing Chen1, Xiao-yu Kang2, Chuan-xi Tang2, Dian-shuai Gao2   

  1. 1 Experiment Teaching Center of Morphology, Xuzhou Medical University, Xuzhou, Jiangsu Province, China;          
    2 Teaching and Research Section of Neurobiology and Anatomy, Xuzhou Medical University, Xuzhou, Jiangsu Province, China
  • Received:2017-06-20 Online:2017-08-15 Published:2017-08-15
  • Contact: Dian-shuai Gao, Ph.D.,gds@xzhmc.edu.cn.
  • Supported by:

    This study was supported by the National Natural Science Foundation of China, No. 81372698.

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摘要：

有文献指出Pitx3基因与Pitx3与GDNF之间的关系尚不明确。实验构建和筛选携带大鼠Pitx3-shRNA的慢病毒表达质粒，并检测在MES23.5多巴胺能神经细胞中敲减Pitx3基因对GDNF转录活性的影响。设计合成3对干扰序列，分别连接于GV102表达载体上。将重组质粒分别转染入MES23.5细胞中，Western blot方法检测Pitx3蛋白表达。最后，将转染效果最好的pitx3 shRNA与携带GDNF启动子区的双荧光素酶报告基因质粒（GDNF-luciferase）共转染至MES23.5细胞。基因测序见合成的序列与Pitx3的3条干扰序列完全一致。荧光倒置显微镜观察见携带Pitx3 shRNA的慢病毒表达质粒的转染效率为40%-50%，并且Western blot结果证实第3条敲减序列对应的Pitx3蛋白表达量最低。双荧光素酶报告基因检测结果见共转染Pitx3 shRNA质粒及GDNF-luciferase质粒组的GDNF的转录活性比单独转染GDNF-luciferase 质粒组的转录活性低。结果证实了实验设计的Pitx3 shRNA干扰序列能够降低多巴胺能神经细胞中Pitx3蛋白表达及GDNF的转录活性。 多巴胺能神经细胞的表型、分化和存活关系密切，但多巴胺能神经细胞中

orcid:0000-0001-8567-0238(Dian-shuai Gao)

关键词: 神经再生, 神经退行性变, 帕金森病, 胶质源性神经营养因子, Pitx3, MES23.5细胞, shRNA, 基因敲除, 质粒, 双荧光素酶报告基因

Abstract:

Pitx3 is strongly associated with the phenotype, differentiation, and survival of dopaminergic neurons. The relationship between Pitx3 and glial cell line-derived neurotrophic factor (GDNF) in dopaminergic neurons remains poorly understood. The present investigation sought to construct and screen a lentivirus expression plasmid carrying a rat Pitx3 short hairpin (sh)RNA and to assess the impact of Pitx3 gene knockdown on GDNF transcriptional activity in MES23.5 dopaminergic neurons. Three pairs of interference sequences were designed and separately ligated into GV102 expression vectors. These recombinant plasmids were transfected into MES23.5 cells and western blot assays were performed to detect Pitx3 protein expression. Finally, the most effective Pitx3 shRNA and a dual-luciferase reporter gene plasmid carrying the GDNF promoter region (GDNF-luciferase) were cotransfected into MES23.5 cells. Sequencing showed that the synthesized sequences were identical to the three Pitx3 interference sequences. Inverted fluorescence microscopy revealed that the lentivirus expression plasmids carrying Pitx3-shRNA had 40–50% transfection efficiency. Western blot assay confirmed that the corresponding Pitx3 of the third knockdown sequence had the lowest expression level. Dual-luciferase reporter gene results showed that the GDNF transcriptional activity in dopaminergic cells cotransfected with both plasmids was decreased compared with those transfected with GDNF-luciferase alone. Together, the results showed that the designed Pitx3-shRNA interference sequence decreased Pitx3 protein expression, which decreased GDNF transcriptional activity.

Key words: nerve regeneration, neurodegeneration, Parkinson’s disease, glial cell line-derived neurotrophic factor, Pitx3, MES23.5 cells, short hairpin RNA, gene knockdown, plasmid, dual-luciferase reporter gene, neural regeneration