Wnt/β-catenin对低氧诱导绿色荧光蛋白小鼠神经干细胞体外增殖的影响

Role of Wnt/β-catenin pathway in proliferation of neural stem cells induced by lower oxygen using green fluorescent proteina-transgenic mouse

目的:体外观察低氧对绿色荧光蛋白(GFP)小鼠神经干细胞(NSCs)增殖的影响,并探讨Wnt/β-catenin通路在此过程中的作用。方法:经低氧(3±2)%O_2和常氧205O_2培养GFP小鼠海马组织NSCs,通过NSCs克隆形成率检测、BrdU掺入法和四唑盐比色法(MTT)法检测细胞增殖情况;脂质体介导法将NSCs转染β-catenin,通过免疫细胞化学染色、Western印迹法检测β-catenin表达情况,MTT法检测转染β-catenin后低氧对NSCs增殖的影响。结果:低氧组细胞克隆球形成率、BrdU阳性细胞数量、NSCs增殖活性、NSCs内β-catenin表达均显著高于常氧组;转染β- catenin的NSCs经低氧培养后,NSCs增殖活性明显高于未转染组。结论:体外低氧培养能促进NSCs增殖和NSCs内β-catenin表达;增加NSCs中β-catenin表达能促进低氧培养下NSCs的增殖。表明Wnt/β-catenin通路具有促进低氧诱导NSCs增殖的作用。

Objective; To observe the effect of lower oxygen on neural stem cells （NSCs） proliferation of the green fluorescence protein （GFP） -transgenic mouse and explore the role of Wnt/β-catenin in this course. Methods; NSCs obtained from the hippocampus of the neonatal mouse were cultured in lower oxygen （3±2）% O2 and traditional oxygen （20%） O2. The proliferation was estimated by clone counts, bromodeoxyuridine （BrdU） uptake and MTT. β-catenin gene was transfected to NSCs by using lipofectamine technique, and the expression of β-catenin in NSCs was detected by immunocytochemical staining and Western blot. The proliferation of transfected NSCs in lower oxygen was estimated by MTT. Results： The yield of clones, BrdU positive cells, the proliferative activity of NSCs and the expression of β-catenin in NSCs were higher in lower O2 compared with those in 20% O2. The proliferative activity of β-catenintransfected NSCs was higher than that of untransfected NSCs in lower oxygen （P 〈 0. 05） . Conclusion： Culturing in lower O2 can promote the proliferation of NSCs and expression of β-catenin in NSCs in vitro ; the increased expression of β-catenin in NSCs can promote the proliferative activity in lower oxygen, suggesting that Wnt/β-catenin signaling may promote the proliferation of NSCs induced by lower oxygen in vitro.