内源性Nurr1对胚胎中脑及前脑前体细胞体外分化的诱导作用

Endogenetic Nurrl for inducing in vitro differentiation of precursors in midbrains and forebrains of embryos

目的:多巴胺神经元的发育是人们较为关注的问题,目前发现的蛋白可能在其发育机制中起着重要作用,研究内源性Nurr1基因表达变化对体外培养大鼠胚胎神经前体细胞分化的诱导作用。方法:实验于2003-05/2004-08在首都医科大学北京神经科学研究所完成。分离13.5d大鼠胚胎中脑及前脑神经前体细胞并进行体外培养,培养基中加入视黄酸及毛喉素诱导3d,采用RT-PCR,WesternBlot及免疫荧光染色的方法观察Nurr1表达的变化及细胞分化;同时,用反义寡核苷酸转染技术阻断细胞Nurr1表达后再进行诱导,并进行同样观察。结果:视黄酸及毛喉素可诱导前体细胞的Nurr1表达,增加约60%,中脑来源神经前体细胞中(tyrosinehydroxylase,TH)阳性细胞占β-tublin-III阳性细胞的比例由原来的(5.32±1.20)%增至(9.01±3.20)%。前脑来源神经前体细胞中TH阳性细胞占β-tublin-III阳性细胞的比例由原来的(1.27±1.20)%增至(5.01±1.50)%。Nurr1反义寡核苷酸作用12h后前体细胞的Nurr1表达被阻断,至72h后重新表达。Nurr1被阻断后,只有少量的前体细胞被诱导为TH阳性细胞。结论:内源性Nurr1的过表达可促进胚胎神经前体细胞向多巴胺能神经元分化。

AIM:The growth of dopamine neur is an hot problem;people have found that Nurr1 plays an important role in the growth mechanism.To study the inducing effect of Nurr1 expression on neural precursors differentiation in rat embryos in vitro was the purpose of this study. METHODES:The experiment was finished in the Institute of Neuroscience of Beijing,Capital University of Medical Sciences from May 2003 to August 2004.The mesencephalic and prosencephalic precursors isolated from rat embryos of 13.5 days were cultured in medium containing retinoic acid(RA) or Forskolin(KF) for three days.The techniques of RT- PCR,Western Blot and immunocytochemistry were used to observe the changes of expression of Nurr1 and cell differentiation.The antisense oligonucleotide was used to block expression of Nurr1 followed by the induction,and then the changes of expression of Nurr1 and cell differentiation were observed. RESULTS:The expression of Nurr1 in cultured precursors induced by RA and FK was increased by about 60% .The ratio of tyrosine hydroxylase(TH) positive cells to β - tublin- III positive cells from mesencephalic precursors was increased from(5.32± 1.20)% to(9.01± 3.20)% , and the ratio of TH positive cells to β - tublin- III positive cells in prosencephalic precursors was increased from(1.27± 1.20)% to(5.01± 1.50)% .The expression of Nurr1 was blocked by antisense oligonucleotide of 12 hours and expressed again after 72 hours.Few of TH positive cells was observed when Nurr1 was blocked by Nurr1 antisense oligonucleotide CONCLUSION:Overexpression of endogenous Nurr1 can accelerate the differentiation of dopaminergic neurons in neural precursors in embryonic brain during development.