人DAT基因的克隆及其对MN9D细胞多巴胺代谢的影响  

Clone of dopamine transporter gene and the effects of overexpression on the transportation of dopamine in MN9D cells

作　　者：周岩[1] 张京钟[1] 赵春礼[1] 赵焕英[1] 徐群渊[1] 

机构地区：[1]首都医科大学神经科学研究所,北京市神经损伤修复重点实验室,北京100069

出　　处：《神经解剖学杂志》2010年第6期573-577,共5页Chinese Journal of Neuroanatomy

基　　金：北京市优秀人才资助项目(2009D0050180000011)

摘　　要：目的:探讨人多巴胺(DA)转运体(dopamine transporter,DAT)基因过表达对单胺类递质代谢的效应。方法:从人胎脑中提取总RNA,RT-PCR方法扩增DAT cDNA片段,重组于pGEM-T-EASY载体中并进行全序列测定。重组真核表达载体pLNCX2-DAT转染MN9D细胞,Western Blot检测DAT的表达,高压液相(high-performance liquid chromatography,HPLC)法检测细胞内外DA含量的变化。结果:RT-PCR方法扩增得到1981bp的cDNA片段,测序结果表明所得到的片段与DAT序列完全一致;Westein Blot证实转染后的MN9D细胞(实验组)内DAT基因表达明显高于对照组(P<0.05);HPLC结果表明实验组中细胞内外的DA含量明显高于对照组(P<0.05)。结论:DAT高表达明显促进DA的循环和利用,为帕金森病的基因治疗提供了理论依据。Objective：To study the effects of dopamine transporter（DAT） overexpression in MN-9D cells on the transportation of dopamine（DA）.Methods：DAT coding sequence was reverse transcripted by RT-PCR from total RNA of human fetal brain,and subcloned into eukaryotic expression vector pLNCX2.Following transfection of pLNCX2-DAT plasmid into MN-9D cells,overexpression of DAT protein was detected with Western Blot.Quantification of DA inside and outside of MN-9D was measured with high-performance liquid chromatography（HPLC）.Results：Reverse transcription of DAT cDNA was 1981 bp.Sequencing results indicated that the fragment fully consistent with the DAT sequence;Western Blot confirmed that the expression of DAT gene in transfected MN90 cells was higher than control group（P0.05）;HPLC results showed that the contentraction of DA was significantly increased inside or outside of the transfected cells（P0.05）.Conclusion：Overexpression of DAT may promote the reuse and inward-transportation of DA and might be potentially use for the gene therapy of Parkinson’s disease.

关 键 词：帕金森病 多巴胺转运体 基因表达 MN9D细胞 

分 类 号：R742.5[医药卫生—神经病学与精神病学]