摘要
目的探讨小鼠海马神经干细胞(NSCs)的体外培养及Wnt/β-catenin信号通路激活剂Wnt3a对体外培养NSCs增殖和分化的影响,以期为脑血管与神经系统疾病的预防与治疗提供新的思路。方法通过无血清悬浮培养法获得新生24 h小鼠海马NSCs,光镜观察NSCs的形态变化,免疫化学染色法鉴定。将NSCs分为正常对照组(sham组),Wnt3a低剂量组(10μg/ml)、Wnt3a中剂量组(25μg/ml)和Wnt3a高剂量组(50μg/ml)。CCK-8法检测各组NSCs增殖活力;流式细胞仪检测各组NSCs的细胞周期; Western blot法检测各组中神经元特异烯醇化酶(NSE)与胶质纤维酸性蛋白(GFAP)的表达变化。结果成功分离、培养小鼠海马NSCs,光镜下呈典型细胞球,巢蛋白(Nestin)阳性。免疫荧光检测显示,NSCs经诱导分化为NSE和GFAP阳性细胞。CCK-8法检测表明,与sham组比较,随着Wnt3a浓度的增加,相应的培养组细胞在450 nm处吸光度值增加,NSCs的增殖活力增加,其促进作用呈剂量依赖性(P <0. 05)。流式细胞仪检测显示,与sham组比较,随着Wnt3a浓度的增加,相应的培养组细胞所处G1期比例降低,(S+G2)期比例升高,NSCs的增殖速率提高(P <0. 05)。Western blot显示,与sham组比较,Wnt3a低、中和高剂量组的NSE蛋白相对表达量提高(P <0. 05),而GFAP蛋白相对表达量降低(P <0. 05)。结论体外成功培养NSCs; Wnt3a激活Wnt/β-catenin信号通路可调控NSCs的增殖,促进其向神经元方向分化,抑制其向星形胶质细胞方向分化。
Objective To investigate the culture of neural stem cells( NSCs) from mouse hippocampus in vitro and the effect of Wnt3a-activated Wnt/β-catenin signaling pathway on NSCs,in order to provide a new strategy for the prevention and treatment of cerebrovascular and neurological diseases. Methods The NSCs were primarily cultured from newborn 24 h mouse hippocampus by serum-free suspension culture. The morphological changes of NSCs were observed by light microscope and identified by immunochemical staining. Then the NSCs were divided into the sham group,Wnt3a low-dose group( 10 μg/ml),Wnt3a middle-dose group( 25 μg/ml) and Wnt3a high-dose group( 50 μg/ml). The viability of NSCs in each group was detected by CCK-8 assay;the flow cytometry was used to detect the cell cycle of NSCs in each group;the expression of NSE and GFAP proteins of NSCs were detected by Western blot in each group. Results NSCs of mouse hippocampus were successfully isolated and cultured. These NSCs appear as cell spheres under light microscopy and express Nestin. NSCs were induced to differentiate into NSE and GFAP positive cells by immunofluorescence detection. Compared with the sham group,the 450 nm absorbance of the cultured cells increased with the increase of Wnt3a concentration,it showed a dose-dependent relationship( P< 0. 05). Compared with the sham group,the proportion of cultured cells in the G1 and( S + G2) phases were decreased with the increase of Wnt3a concentration( P < 0. 05). The relative expression of NSE protein in Wnt3a low-dose group,Wnt3a mid-dose group and Wnt3a high-dose group were significantly higher than that in sham group( P < 0. 05),but the relative expression of GFAP protein was significantly lower than that in sham group( P <0. 05). Conclusion NSCs were successfully isolated and cultured,Wnt3a could significantly enhance the proliferation of NSCs,promote its differentiation into neurons and inhibit its differentiation into astrocytes by activating Wnt/β-catenin signaling pathway.
作者
张先虎
朱俊德
龙婷婷
王俊婕
康朝胜
Zhang Xianhu;Zhu Junde;Long Tingting(Dept of Anatomy, School of Basic Medicine, Guizhou Medical University, Guiyang 550025)
出处
《安徽医科大学学报》
CAS
北大核心
2019年第1期44-49,共6页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金项目(编号:81660243)
贵州省科技厅社会发展科技计划项目(编号:黔科合SY字[2015]3041号)
贵州省科技厅与贵州医科大学联合项目(编号:黔科合LG字[2012]028号)
