Ethanol Affects Differentiation-Related Pathways and Suppresses Wnt Signaling Protein Expression in Human Neural Stem Cells
Ethanol Affects Differentiation-Related Pathways and Suppresses Wnt Signaling Protein Expression in Human Neural Stem Cells
复制标题
DOI：
10.1111/j.1530-0277.2011.01682.x
复制
发表时间：
2012-05-01
影响因子：
3.2
通讯作者：
Lyman, William D.
中科院分区：
文献类型：
--
作者：
Vangipuram, Sharada D.;Lyman, William D.
背景：胎儿产前暴露于乙醇（EtOH）可能是致畸的。我们之前的研究表明，EtOH可以改变人神经干细胞（NSC）的细胞命运。由于Wnt信号在胎儿大脑发育中起重要作用，我们假设EtOH在NSC分化过程中抑制Wnt信号蛋白的表达，从而导致胎儿酒精谱系障碍。方法：用有丝分裂培养基培养人脑胚NSC诱导神经球，将其分离成单细胞悬液，用于所有实验。等量的NSC在赖氨酸/层粘连蛋白包被板上，在含有0、20或100 mM EtOH的分化培养基中培养96小时。从含有0或100 mM EtOH的样品中分离总mRNA，并通过Oligo GEArray技术检测与神经发生和NSC分化相关的263个基因的表达变化。基因变化的生物学影响使用系统生物学方法与途径表达软件和KEGG数据库进行估计。基于所鉴定的通路，Western blot分析Wnt蛋白（Wnt3a和Wnt5a）、Wnt受体复合物蛋白（p-LRP6、LRP6、DVL2和DVL3）、Wnt拮抗剂NKD-2和下游Wnt蛋白（β -catenin、tyrr -p- gsk3 β、Ser-p-GSK3 β）的表达。结果：在所检测的263个基因中，有22个基因在NSC分化过程中受EtOH的影响呈上升或下降趋势。这些基因与5种途径/细胞过程相关：轴突引导；刺猬信号;及信号;细胞粘附分子；和Wnt信号。与对照组相比，20和100 mM浓度的EtOH均抑制了Wnt3a和Wnt5a、受体复合物蛋白p-LRP6、LRP6和DVL2以及细胞质蛋白Ser-p-GSK3 β和β -catenin的表达。NKD-2和DVL3的表达保持不变，活性tyrr -p- gsk3 β的表达显著增加。结论：EtOH可显著改变人NSC分化过程中神经分化通路相关基因表达，抑制Wnt信号蛋白表达。
Background: Prenatal exposure of the fetus to ethanol (EtOH) can be teratogenic. We previously showed that EtOH alters the cell fate of human neural stem cells (NSC). As Wnt signaling plays an important role in fetal brain development, we hypothesized that EtOH suppresses Wnt signaling protein expression in differentiating NSC and thereby contributes to fetal alcohol spectrum disorder. Methods: NSC isolated from fetal human brains were cultured in mitogenic media to induce neurospheres, which were dissociated into single-cell suspensions and used for all experiments. Equal numbers of NSC were cultured on lysine/laminin-coated plates for 96 hours in differentiating media containing 0, 20, or 100 mM EtOH. Total mRNA was isolated from samples containing 0 or 100 mM EtOH and changes in expression of 263 genes associated with neurogenesis and NSC differentiation were determined by Oligo GEArray technology. The biological impact of gene changes was estimated using a systems biology approach with pathway express software and KEGG database. Based on the pathways identified, expression of Wnt proteins (Wnt3a and Wnt5a), Wnt-receptor complex proteins (p-LRP6, LRP6, DVL2, and DVL3), Wnt antagonist Naked-2 (NKD-2), and downstream Wnt proteins (beta-catenin, Tyr-p-GSK3 beta, Ser-p-GSK3 beta) were analyzed by Western blot. Results: Of the 263 genes examined, the expressions of 22 genes in differentiating NSC were either upwardly or downwardly affected by EtOH. These genes are associated with 5 pathways/cellular processes: axon guidance; hedgehog signaling; TGF-beta signaling; cell adhesion molecules; and Wnt signaling. When compared to controls, EtOH, at both 20 and 100 mM concentrations, suppressed the expression of Wnt3a and Wnt5a, receptor complex proteins p-LRP6, LRP6 and DVL2, and cytoplasmic proteins Ser-p-GSK3 beta and beta-catenin. Expression of NKD-2 and DVL3 remained unchanged and the expression of active Tyr-p-GSK3 beta increased significantly. Conclusions: EtOH can significantly alter neural differentiation pathway-related gene expression and suppress Wnt signaling proteins in differentiating human NSC.