Wnt信号通路分子在大鼠神经干细胞分化过程中的表达

Expression profiles of Wnt signaling molecules in differentiation process of rat neural stem cells

摘要:

目的 观察Wnt信号通路主要分子[β-连环蛋白（β-catenin）和糖原合酶激酶-3β（GSK-3β）]在神经干细胞（NSCs）体系的表达及其在增殖和分化过程中的变化，探讨其在神经干细胞分化中可能的调控作用。方法 采用原代培养方法，自14～16d大鼠胚胎大脑皮后获得含大量细胞团的NSCs体系。应用含10%胎牛血清培养液诱导NSCs分化。Western印迹检测NSCs在加入胎牛血清12，24，48和72h后，β-catenin与GSK-3β在分化过程中的动态变化。结果 免疫细胞化学染色显示NSCs细胞团及散在的单个细胞多呈巢蛋白（nestin）阳性，显示NSCs分化过程中两者存在持续表达，β-catenin分子表达呈现逐渐减少趋势，而GSK-3β分子表达则呈现逐渐增加趋势。结论 提示Wnt信号通路与NSCs的增殖和分化过程密切相关，呈现出由活跃到逐步抑制的过程，为研究Wnt信号通路参与NSCs增殖分化调控的机制提供了载体。

Abstract:

Objective To determine the expression profiles of main molecules in Wnt signal pathway, β-catenin and glycogen synthase kinase-3β (GSK-3β), in the proliferation and differentiation process of neural stem cells (NSCs), and to investigate the possible regulating role of these molecules in the process. Methods The NSCs were isolated and primarily cultured from rat fetus at an embryonic age of 14 to 16d according to reported methods. At 12 to 24h after cultured in the medium containing 10% fetal bovine serum (FBS), the cells attached and grew gradually. Western blotting was used to measure the protein levels of β-catenin and GSK-3β in the cells treated in the FBS medium or control after 12, 24, 48 and 72 hours treatment. Results Immunofluorescence assay indicated that the cultured neurospheres or single cells were positive with nestin. Western blotting results revealed a continuous expression of β-catenin and GSK-3β in the differentiation of the cultured NSCs. β-catenin was in a gradual decreasing tendency, whereas GSK-3β showed an opposite change. Conclusions Wnt signaling pathway is involved in the proliferation and differentiation of NSCs, and is active first and then gradually inhibited. These confirm the NSCs biological property of the culturing cells and provide a model for studying the role of Wnt signaling molecules.