Ⅱ型囊泡单胺转运体基因过表达对多巴胺含量影响的体外研究

EFFECT OF OVEREXPRESSING VESICULAR MONOAMINE TRANSPORTER-2 GENE ON THE CONTENTS OF DOPAMINE IN VITRO

目的 获得人Ⅱ型囊泡单胺转运体 (VMAT2 )基因 ,转染至MN9D细胞 ,通过检测细胞内外单胺类递质含量的变化评价VMAT2 基因促进单胺类递质循环利用的效应。 方法 从人胎脑中提取总RNA ,RT PCR方法扩增目的cDNA片段并将其重组于pGEM Easy T载体中 ,进行全序列测定。重组真核表达载体pBK RSV VMAT2 ,转染MN9D细胞 ,免疫细胞化学检测VMAT2 的表达 ,HPLC检测细胞内外单胺类递质含量的变化。 结果 RT PCR扩增到 16 37bp的带有Kozak序列的cDNA片段。原位杂交证实pAAV VMAT2 能在COS7细胞表达 ;免疫组织化学证实转基因的MN9D细胞 (实验组 )VMAT2 免疫着色明显高于对照组。高效液相色谱结果表明 ,实验组中细胞内外的多巴胺含量均升高 (P <0 0 1) ,细胞外HVA、DOPAC降低 (P <0 0 5 ) ,而胞内升高 (P <0 0 1)。 结论 克隆的VMAT2cDNA片段可在真核细胞中表达 ,该基因的过表达可促进多巴胺的循环利用 ,有望用于帕金森病的基因治疗。

Objective To increase the efficiency of transporting dopamine distributed in cytoplasm. Methods Human VMAT-2 gene derived from midbrain of aborted fetus was cloned and an expressing vector pBK-RSV-VMAT-2 was constructed. This vector was then infected into the MN9D cells. The genetically modified MN9D cells expressing VMAT-2 were detected by in situ hybridization and immunocytochemistry. The levels of dopamine(DA) and its metabolites, dihydroxyphenyl acetic acid (DOPAC) and homovanillic acid (HVA) in the media and cell pellets were determined by HPLC. Results The expression of VMAT-2 in genetically modified MN9D cells significantly increase compared with MN9D cells(P<0.05) for control. The levels of DOPAC and HVA decreased in the media of genetically modified MN9D cells(P<0.05). However, the levels of dopamine increased in the medium and cell pellets of genetically modified MN9D (P<0.05) compared with MN9D cells.Conclusion The VMAT2 can relatively increase the efficiency of recirculating dopamine in the genetically modified MN9D cells in vitro and VMAT-2 gene may be a promising target gene for Parkinson disease (PD) gene therapy.