低氧促进神经干细胞向多巴胺能神经元分化

神经干细胞（neural stem cells,NSCs）作为具有多向分化潜能的神经前体细胞,被广泛应用于细胞移植等研究,而低氧不但调节干细胞的体外增殖,在干细胞分化中也具有重要的作用。本文着重探讨了低氧对NSCs分化的调节作用。采用Wistar孕大鼠（E13．5d）,分离胚胎中脑NSCs,加入无血清DMEM／F12培养液（含20ng／mL EGF、20ng／mL bFGF、1％ N2和B27）,3～5d后传代,细胞培养至第三代进行诱导分化,分别在低氧（3％O2）和常氧（20％O2）条件下诱导分化3d,然后在常氧条件下分化成熟5～7d（DMEM／F12含1％FBS、N2和B27）后进行检测。Nestin、NeuN以及TH免疫组织化学鉴定NSCs;流式细胞术分析测定NSCs向TH阳性神经元方向的分化;高效液相色谱测定细胞培养上清液中多巴胺（dopamine,DA）含量。结果显示,分离培养的NSCs均为nestin阳性细胞;低氧可明显促进NSCs向神经元方向的分化;TH阳性神经元比例在常氧和低氧组分别为（10．25±1．03）％和（19．88±1．44）％。NSCs诱导分化7d后,低氧组细胞培养上清液中DA浓度明显增加,约为常氧组的2倍（P〈0．05,n=8）。上述结果表明,3％低氧可促进NSCs向神经元方向,特别是向DA能神经元方向分化。这为NSCs应用于临床治疗帕金森病提供了基础。

Hypoxia promotes the differentiation of neural stem cells into dopaminergic neurons

Neural stem cells (NSCs) are multipotent and widely used in many research fields such as transplantation. Hypoxia not only improves the proliferation of various stem cells in vitro but also plays an important role in the differentiation of stem cells. The aim of the present study was to investigate the effect of hypoxia on the differentiation of NSCs. NSCs were isolated from the midbrain of embryonic Wistar rats (E13.5d), and cultured in serum-free DMEM/F12 medium (containing 20 ng/mL EGF, 20 ng/mL bFGF, 1% N2 and B27). The neurospheres were passaged every 3-5 d, and the third generation of NSCs was used for the following experiments. NSCs were induced under normoxia (20% O2) and hypoxia (3% O2), respectively, for 3 d, and then differentiated under normoxia for 5-7 d (DMEM/F12 medium containing 1% FBS, N2 and B27). Immunohistochemistry of nestin, NeuN and TH was used for NSC identification and differentiation assay. The number of TH-positive cells was evaluated by flow cytometry. Dopamine (DA) content in the supernatant of culture medium was detected by HPLC. The results showed that NSCs could self-renew and were all nestin-positive. NSCs under hypoxic condition differentiated more neurons than those under normoxic condition. The percentage of TH-positive cells differentiated from NSCs under normoxia and hypoxia was (10.25+/-1.03) % and (19.88+/-1.44) %, respectively. In addition, DA content in the supernatant of culture medium in hypoxia group increased significantly, about twice of that in normoxia group (P<0.05, n=8). The results demonstrate that hypoxia (3% O2) promotes the differentiation of NSCs into neurons, especially dopaminergic neurons. It is suggested that hypoxia may be a potential clinical tool to treat Parkinson's disease.