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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 investigate the effect of dopamine transporter (DAT) gene overexpression on monoamine metabolites. Methods: Total RNA was extracted from human fetal brain. The cDNA fragment of DAT was amplified by RT-PCR and sequenced in pGEM-T-EASY vector. The recombinant eukaryotic expression vector pLNCX2-DAT was transfected into MN9D cells, the expression of DAT was detected by Western Blot and the changes of intracellular DA content were detected by high-performance liquid chromatography (HPLC). Results: The 1981 bp cDNA fragment was amplified by RT-PCR. The sequencing results showed that the obtained fragment was completely consistent with the DAT sequence. Westein Blot confirmed that the expression of DAT gene in MN9D cells (experimental group) was significantly higher than that in the control group P <0.05). The HPLC results showed that the content of intracellular DA in the experimental group was significantly higher than that in the control group (P <0.05). Conclusion: The high expression of DAT significantly promotes the circulation and utilization of DA, which provides a theoretical basis for the gene therapy of Parkinson’s disease.