GDF11对小鼠神经干细胞增殖及TGF-β／Smads、Wnt／β-连环蛋白信号通路的影响

Growth differentiation factor 11 promotes proliferation of mouse neural stem cells and activates both transforming growth factor-β/Smads and Wnt/β-Catenin signal pathways

目的探讨生长分化因子11（GDF11）对小鼠神经干细胞增殖及转化生长因子-β（TGF—β）／Smads、Wnt／β-连环蛋白信号通路关键蛋白表达的影响。方法分离培养及诱导分化孕14dCD1胎鼠大脑侧脑室下区的神经干细胞，采用免疫荧光染色鉴定神经干细胞特异性蛋白巢蛋白、SOX2，以及在诱导分化后鉴定神经元标志物神经核抗原（NeuN）和星型胶质细胞标志物胶质纤维酸性蛋白（GFAP）。取第3代神经干细胞分为实验组和对照组，实验组添加GDF11（终浓度40ng／mL）．对照组添加等体积完全培养液。采用EdU法检测2组细胞增殖情况，采用Westem blotting于培养1h、6h时检测2组细胞Smad2／3、磷酸化（p）-Smad2／3、Smad4、β-连环蛋白表达。结果免疫荧光染色显示90％以上细胞呈巢蛋白和SOX2阳性，诱导分化后部分细胞呈NeuN或GFAP阳性。细胞增殖实验显示：与对照组EdU阳性细胞比例（0．24±0．03）相比，实验组EdU阳性细胞比例（0．34±0．05）明显提高，差异有统计学意义（P〈0．05）。Western blotting结果显示：实验组P—Smad2／3、Smad4和β-连环蛋白表达在培养1h、6h时均明显高于对照组，差异均有统计学意义（P〈0．05）。结论GDF11可在体外促进小鼠神经干细胞增殖，其机制可能与TGF-β／Smad、Wnt／β-连环蛋白信号通路激活有关。

Objective To investigate the effects of growth differentiation factor 11 （GDF11） on proliferation of mouse neural stem cells （NSCs） and expression levels of transforming growth factor （TGF）-β/Smads and Wnt/β-Catenin signal key proteins. Methods NSCs, derived from the subventricular zone of El4 d CD1 mice, were cultured and induced differentiation; specific proteins nestin and SOX2 were confirmed by immunofluorescence assay. Neuron marker nucleus antigen （NeuN） and astrocyte marker glial flbrillary acidic protein （GFAP） were identified by immunofluorescent staining. The cells of third generation in their exponential phase were chosen and randomly divided into experimental group （adding GDF11 to make the final concentration as 40 ng/mL） and control group （adding equal amount of culture fuid）. The proliferation of the cells in the two groups was detected by 5-ethynyl-2＇-deoxyuridine （EdU） kits and protein expressions of Smad2/3, phosphorylated （p）-Smad2/3, Smad4 and β-Catenin were measured by Western blotting one and 6 h after treatment. Results Round and bright cells suspended in culture medium were observed through optical microscope. Immunofluorescence assay showed that over 90% cells expressed both nestin and SOX2, and some of them expressed NeuN or GFAP. EdU proliferation test showed that the percentage of EdU positive cells in the experimental group （0.34±0.08） was significantly higher than that in the control group （0.24±0.03, P〈0.05）. Western blotting showed that the expression levels ofp-Smad2/3, Smad4 and β-Catenin were significantly increased one and 6 h after treatment as compared with those in the control group （/9〈0.05）. Conclusion GDF11 can promote the proliferation of NSCs in vitro and probably is on account of activating TGF-β/Smads and Wnt/β-Catenin signal pathways.