帕金森病基因治疗的理论:VMAT_2基因的表达及其对MN9D细胞多巴胺代谢的影响

Effect of vesicular monoamine transporter-2 gene expression on the metabolites of dopamine in MN9D cells

目的:获得人Ⅱ型囊泡单胺转运体(vesicularmonoaminetransporter-2,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1637bp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;免疫组化证实转基因的MN9D细胞(实验组)VMAT2免疫着色明显高于对照组。HPLC结果表明实验组中细胞内外的多巴胺含量分别升高至(227.2±20.8)%和(302.6±15.7)%(P<0.01),而细胞外HVA、DOPAC分别降低至(48.1±5.3)%和(55.7±4.9)%(P<0.05),细胞内HVA,DOPAC分别升高至(332.1±28.2)%和(1176.8±82.5)%(P<0.01)。结论:克隆了VMAT2cDNA片断,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。

AIM:To evaluate the efficiency of vesicular monoamine transporter-2(VMAT2) in promoting the circulating usage of monoamine transmitters by the detection of the contents of monoamine transmitters inside and outside cells. METHODS:Total RNA was extracted from human fetal brains.cDNA fragments were a mplified with RT-PCR technique and recombined in PGEM-Easy-T vector for th e complete sequence arrangement.Recombinant eukaryocyte expression vector pBK-RSV-VMAT2 was used to transfect MN9D cells.The expression of VMAT2 was detecte d by immunocytochemistry and the contents of monoamine transmitters were measure d with high pressure liquid chromatog raphy(HPLC). RESULTS:After amplification with RT-PCR,a 1637-bp Kozak sequence was obta ined.In situ hybridization detected that pAAV-VMAT2 expressed in COS7 cells.Im munohistochemistry showed that transgenic MN9D cells in the experimental group w ere stained more obviously than that in the control group.The findings of HPLC p resented that the contents of intracellular and extracellular dopamine were incr eased to(227.2± 20.8)% and(302.6± 15.7)% respectively(P< 0.01),while the c ontents of extracellular homovanillic acid(HVA) and dihydroxyphenyl acetic acid( DOPAC) were decreased to(48.1± 5.3)% and(55.7± 4.9)% (P< 0.05) respectively ,and the contents of intracellular HVA and DOPAC was respectively increased to(3 32.1± 28.2)% and(1 176.8± 82.5)% (P< 0.01). CONCLUSION:VMAT2 can relatively increase the efficiency of recirculation of d opamine and VMAT2 gene may be a promising target gene on gene therapy for Parkin son's disease.