补阳还五汤含药血清对体外缺氧损伤神经干细胞增殖及Notch信号通路相关因子mRNA表达的影响

Effects of Buyang Huanwu Decoction serum on the expression of Notch signaling pathway related factors mRNA in neural stem cells after hypoxia injure in vitro

目的:探讨补阳还五汤含药血清对体外缺氧损伤神经干细胞(NSCs)增殖及Notch信号通路相关因子m RNA表达的影响。方法:体外分离培养小鼠胚胎NSCs,取第3代悬浮生长的NSCs随机分组,空白组予以无血清条件培养基常氧培养,其余3组分别给予缺氧4h、缺氧4h加10%正常血清和缺氧4h加10%补阳还五汤含药血清干预;培养第5天观察细胞增殖情况,同时提取细胞总RNA,Real-time PCR检测Notch通路4个关键节点因子Notch-1、Hes-1、Neurogenin-1(Ngn-1)和Delta-like-1(Dll-1)m RNA相对表达水平。结果:正常生长的NSCs Notch-1、Hes-1 m RNA高表达,Ngn-1、Dll-1 m RNA低表达;缺氧4h后NSCs克隆球数减少(P<0.05),Notch-1、Hes-1 m RNA下调(P<0.05),Ngn-1、Dll-1 m RNA上调(P<0.05);10%补阳还五汤含药血清能显著上调缺氧NSCs Notch-1、Hes-1、Ngn-1 m RNA以及下调Dll-1 m RNA的表达水平,增加缺氧NSCs克隆球数,与10%正常血清相比差异显著(P<0.05)。结论:调节NSCs Notch信号通路相关因子Notch-1、Hes-1、Ngn-1、Dll-1 m RNA的表达水平可能是补阳还五汤促进缺氧损伤后NSCs增殖的机制。

Objective： To investigate the effects of Buyang Huanwu Decoction serum on the expression of Notch signaling pathway related factors mRNA in neural stem cells（NSCs） after hypoxia injure in vitro. Methods： The embryos NSCs were isolated from fetal mouse brain and cultured in vitro, and the third generation of suspended growth of the neural spheres were randomly divided into four groups. NSCs of blank group were treated with serum free conditioned culture medium and normal oxygen, and the other three groups were given with hypoxia for 4h, hypoxia for 4h and 10% normal serum intervention as well as lack of oxygen for 4h plus 10% Buyang Huanwu Decoction serum intervention respectively. On the fifth day, the proliferation of cells were observed. Total RNA was extracted from NSCs, and Real-time PCR was used to detect the relative expression level of 4 key factors mRNA of Notch pathway： Notch-1, Hes-1, Neurogenin-1（Ngn-1） and Delta-like-1（Dll-1）. Results： Notch-1, Hes-1 mRNA in normal growth NSCs were high expressed and Ngn-1, Dll-1 were low expressed. The number of NSCs clones decreased after hypoxia 4h（P〈0.05）, along with suppression of Notch-1, Hes-1 expression and promotion of Ngn-1, Dll-1 expression（P〈0.05）. 10% Buyang Huanwu Decoction serum could significantly raise the expression of Notch-1, Hes-1, Ngn-1 mRNA and reduce the Dll-1 mRNA in mouse embryos NSCs as well as increase the number of NSCs clones. What is more, compared with 10% normal serum, the difference was significant（P〈0.05）. Conclusion： Buyang Huanwu Decoction can regulate the expression of Notch signaling pathway related factors Notch-1, Hes-1, Ngn-1 and Dll-1 mRNA, which is probably the mechanism on promoting neurogenesis after brain hypoxia injury.