目的 探讨Wnt/β-catenin信号传导在高压氧(HBO)治疗新生大鼠缺氧缺血性脑损伤(HIBD)中的作用.方法 将新生SD大鼠神经干细胞(NSCs)随机分为对照组和8个处理组,处理组分别与正常脑组织匀浆及HIBD大鼠脑组织匀浆共培养,以模拟正常及HIBD时脑内微环境,通过电穿孔方法将β-catenin siRNA和阴性对照质粒转染预先处理过的NSCs,再给予HBO处理.运用免疫细胞组织化学方法观察HBO对NSCs分化的影响；Western blot、RT-PCR法观察β-catenin下游基因Ngn1和骨形成蛋白(BMP4)的表达.结果 HBO可促进转染阴性对照质粒的NSCs (ncNSCs)分化为神经元和少突胶质细胞,抑制ncNSCs向星形胶质细胞分化；HIBD及正常大鼠脑组织匀浆均可促进ncNSCs分化为神经元及少突胶质细胞,抑制其向星形胶质细胞分化,HIBD大鼠脑组织匀浆的作用强于正常脑组织匀浆,且HBO可加强这一效果；转染β-catenin siRNA后,NSCs向神经元分化减少,向星形胶质细胞分化增加,这一作用可被HBO减弱,且不可逆转；HBO促进NSCs分化为神经元的同时,Ngn1蛋白和mRNA的表达增加,β-catenin siRNA促进NSCs分化为星形胶质细胞的同时,BMP4蛋白和mRNA的表达增加.结论 HBO可促进离体HIBD大鼠NSCs分化为神经元和少突胶质细胞而抑制其向星形胶质细胞分化；HBO促进离体大鼠NSCs分化为神经元与β-catenin的激活有关；HBO是通过激活β-catenin上调Ngn1的表达促进NSCs分化为神经元,通过下调BMP4的表达减少NSCs分化为星形胶质细胞；BMP4和Ngn1在大鼠NSCs的增殖分化中起重要的协调作用,Ngn1可能是一种潜在的神经元促成剂.%Objective To investigate the role of Wnt/β-catenin signaling pathway in hyperbaric oxygen (HBO) therapy for hypoxic-ischemic brain damage(HIBD).Methods The neural stem cells (NSCs) of neonatal rats were randomly divided into control group and 8 treatment groups,then the treatment groups were cultured in the supernatant of brain homogenate to simulate micro-environment of HIBD and normal brain respectively.Through electroporation β-catenin siRNA and negative control plasmid was transfected into NSCs respectively,then HBO therapy was performed.Immunocytochemical staining was performed simultaneously in the 9 groups of NSCs on precoated chamber slides to detect the differentiation of NSCs.Quantitative reverse transcriptase(RT)-PCR was used to detect the relative content of Ngn1 mRNA and bone morphogenetic protein(BMP4) mRNA in the of NSCs.Western blot was used to detect the relative content of Ngn1 protein and BMP4 protein in the NSCs.Results In vitro,HBO alone promoted NSCs infected with negative control siRNA (ncNSCs) to differentiate into neurons and oligodendrocytes and depressed astrogliosis ; HIBD or normal brain tissue extract cultures promoted ncNSCs to differentiate into neurons and oligodendrocytes but depressed astrogliosis,and the effect of HIBD brain extract cultures was superior to the latter and could be further increased by HBO; β-catenin siRNA decreased the NSE-positive neurons and increased glial fibrillary acidic protein-positive astrocytes in the siNSCs (NSCs infected with β-catenin siRNA) in vitro,the effect could not be inversed by HBO,but could be alleviated; HBO increased the level of Ngn1 mRNA and Ngn1 protein,decreased BMP4 mRNA and BMP4 protein of ncNSCs,transfection of β-catenin siRNA could down-regulate the expression of Ngn1 mRNA and upregulate BMP4 mRNA of NSCs in vitro respectively.Conclusions HBO can promote NSCs cultured with HIBD brain extract cultures to differentiate into neuronal or Oligodendrocyte,and inhibit them to differentiate into astrocytes.HBO therapy promotes the proliferation of NSCs in vitro,an effect which is correlated with β-catenin protein.HBO therapy can promote neurogenesis by β-catenin-induced activated Ngn1,and repress astrocytogenesis by β-catenin-induced down-regulated BMP4.There are potential cooperative actions of BMP4 and Ngn1 on differentiating rat NSCs in cerebral ischemic brain.The ability of Ngn1 to promote neurogenesis may allow Ngn1 to act as a potent neuronal commitment factor.
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