Effects of Nurr1 down-regulation on the expression of tyrosine hydroxylase and neurite extension in dopaminergic cells
- Author:
- No author available
- Journal Title:
- ACTA PHYSIOLOGICA SINICA
- Issue:
- 4
- DOI:
- 10.3321/j.issn:0371-0874.2006.04.009
- Key Word:
- 核受体相关因子1;酪氨酸羟化酶;RNA干扰;帕金森病;发育;神经突起
Abstract： 将合成的核受体相关因子1(nuclear receptor-relatedfactor 1,Nurr1)特异性短发夹寡核苷酸(small-hairpin RNA,shRNA)序列插入真核表达载体pSilenCircle(pSC),构建Nurr1基因特异性shRNA真核表达载体,转染体外培养多巴胺能神经前体细胞系MN9D,分别采用实时荧光定量PCR和Western blot方法检测其对MN9D细胞内源Nurr1的干扰作用及其对酪氨酸羟化酶(tyrosine hydroxylase,TH)表达的影响,并在倒置显微镜下观察MN9D细胞神经突起生长的情况,探讨Nurr1 shRNA表达载体对多巴胺能细胞表型标记物TH和以神经突起延长为特征的细胞成熟的影响.结果表明,脂质体组细胞和转染阴性对照质粒的MN9D细胞内Nurr1、TH的表达正常,而转染Nurr1 shRNA真核表达载体(pSC-N1和pSC-N2)的MN9D细胞内Nurr1和TH的mRNA水平明显降低,Nurr1 mRNA的下降率分别为62.3%和45.6%,TH mRNA的下降率分别为76.3%和62.6%.同时Nurr1和TH蛋白的表达亦明显下调,Nurr1蛋白的下降率分别为57.4%和72.0%,TH蛋白的下降率分别为79.1%和70.1%.另外,转染Nurr1 shRNA真核表达质粒的MN9D细胞神经突起延长有所减少,但是与正常细胞无明显差异.结果提示:Nurr1 shRNA真核表达载体能显著下调MN9D细胞内源Nurr1和TH mRNA和蛋白的表达,同时可能对MN9D细胞的神经突起延长有一定的抑制作用.Nurr1 shRNA表达载体的成功构建为多巴胺能神经元发育以及帕金森病相关基因的功能研究奠定了基础.
- [1]杨秋慧,杨慧,赵焕英,等.Nurr1在MN9D细胞中的过表达及其对酪氨酸羟化酶的影响[J].2003,(3).
- [2]吴云成,赵永波,刘文文.Nurr1基因shRNA表达载体的构建及其鉴定[J].2005,(6).
- [3]张伟,曹越,张昱,等.RNA干扰沉默低氧诱导因子-1α对低氧下大鼠肺动脉平滑肌细胞增殖的影响[J].2006,(1).
- [4]吴云成,赵永波.RNA干扰及其在神经变性性疾病研究中的应用[J].2004,(4).
- [5]Kim YC,Chung HY,Lee SH,etc.Dopaminergic neuronal differentiation from rat embryonic neural precursors by Nurr1 overexpression.[J].2003,85(6).
- [6]A, Fire,S, Xu,M K, Montgomery,etc.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.[J].1998,391(6669).
- [7]Haque ME,Yang LC,Beal MF,etc.Inactivation of Drosophila DJ-1 leads to impairments of oxidative stress response and phosphatidylinositol 3-kinase/Akt signaling[J].2005,102(38).
- [8]S O, Castillo,J S, Baffi,M, Palkovits,etc.Dopamine biosynthesis is selectively abolished in substantia nigra/ventral tegmental area but not in hypothalamic neurons in mice with targeted disruption of the Nurr1 gene.[J].1998,11.
- [9]Hermanson E,Wallen A,Joseph B,etc.Induction of cell cycle arrest and morphological differentiation by Nurr1 and retinoids in dopamine MN9D cells.[J].2001,276(46).
- [10]Panisset M,Han F,Racacho L,etc.Translated mutation in the Nurr1 gene as a cause for Parkinson's disease.[J].2006,21(7).
- [11]Åsa Wallén,Rolf H. Zetterström,Ludmila Solomin,etc.Fate of Mesencephalic AHD2-Expressing Dopamine Progenitor Cells in Nurr1 Mutant Mice[J].1999,253(2).
- [12]R H, Zetterstr?m,L, Solomin,L, Jansson,etc.Dopamine neuron agenesis in Nurr1-deficient mice.[J].1997,276(5310).
- [13]Xu PY,Liang R,Jankovic J,etc.Association of homozygous 7048G7049 variant in the intron six of Nurr1 gene with Parkinson's disease.[J].2002,58(6).
- [14]Choi JE,Cho SL,Hwang O,etc.Construction of a vector generating both siRNA and a fluorescent reporter: a siRNA study in cultured neurons[J].2004,18(1).
- [15]Natt F,Hoyer D,Cryan JF,etc.Neurochemical and behavioral consequences of widespread gene knockdown in the adult mouse brain by using nonviral RNA interference.[J].2004,101(49).
- [16]Le WD,Xu P,Jankovic J,etc.Mutations in NR4A2 associated with familial Parkinson disease.[J].2003,33(1).
- [17]Nishimura I,Yang Y,Takahashi R,etc.Parkin suppresses dopaminergic neuron-selective neurotoxicity induced by Pael-R in Drosophila.[J].2003,37(6).
- [18]Saucedo-Cardenas O,Le WD,Quintana-Hau JD,etc.Nurr1 is essential for the induction of the dopaminergic phenotype and the survival of ventral mesencephalic late dopaminergic precursor neurons.[J].1998,95(7).
- [19]Simmons DL,Sears RM,Georgescu D,etc.Local gene knockdown in the brain using viral-mediated RNA interference.[J].2003,9(12).
- [20]Zheng K,Heydari B,Simon DK.A Common NURR1 Polymorphism Associated With Parkinson Disease and Diffuse Lewy Body Disease.[J].2003,60(5).
- [21]H K, Choi,L A, Won,P J, Kontur,etc.Immortalization of embryonic mesencephalic dopaminergic neurons by somatic cell fusion.[J].1991,552(1).