同型半胱氨酸对体外大鼠神经干细胞增殖及Notch信号通路相关基因表达的影响

Effects of Homocysteine on Neural Stem Cell Proliferation and Genes Related Notch Signaling in Vitro

目的研究同型半胱氨酸(Hcy)对体外大鼠神经干细胞(NSCs)增殖及Notch信号通路相关基因Notch1和Hes1 mRNA表达的影响。方法采用无血清培养原代培养新生大鼠NSCs,细胞分为对照组(Hcy-C)、Hcy低剂量组(Hcy-L)、Hcy中剂量组(Hcy-M)、Hcy高剂量组(Hcy-H)。采用免疫组化法检测Nestin、β-tubulinⅢ及GFAP的表达对NSCs进行鉴定,MTT法检测细胞增殖情况,Real-time PCR检测Notch1、Hes1基因mRNA表达。结果无血清培养条件下,所培养细胞形成大量呈Nestin抗原阳性细胞组成的神经球;诱导分化6 d后,形成β-tubulinⅢ表达呈阳性的神经元和GFAP表达呈阳性的星形胶质细胞;Hcy干预组细胞活力低于对照组,且有剂量依赖性(P<0.05)。Hcy干预组Hes1 mRNA表达低于对照组(P<0.05)。Hcy-H组Notch1 mRNA表达低于其他3组(P<0.05);Hcy-M组Notch1mRNA表达低于对照组和低剂量组(P<0.05)。结论 Hcy能抑制大鼠新生鼠NSCs的增殖,可能与下调Notch信号通路中Notch1和Hes1 mRNA表达有关。

Objective To explore the effects of homocysteine （Hcy） on neural stem cell （NSC） proliferation and the mRNA expression level of Notchl and Hesl related Notch signaling pathway from neonatal rats in vitro. Methods NSCs from neonatal rats were cultured by serum-free culture method in vitro. Cells were divided into four groups： control group （Hcy-C）, low dose Hcy （Hcy-L） group, middle dose Hcy （Hcy-M） group and high dose Hcy （Hcy-H） group. NSCs were identified by immunofluorescent staining using the antibodies against Nestin, β-tubulin m and GFAP. The proliferation ability of NSCs was detected by MTT. The mRNA expressions of Notchl and Hesl were detected by Real-time PCR. Results In the serum free suspension medium, neurospheres that consisted of a great number of nestin-positive cells were found. β-tubulin m positive neurons and GFAP positive astrocytes were detected by immunofluorescence staining on the 6 th day of cell induction. MTT assay showed that the cell viability was significantly lower in three Hcy treatment groups than those of control group （P 〈 0.05）. And the effect of concentration-dependent was observed. The results of RT-PCR showed that mRNA expression of Hesl was significantly lower in three Hcy treatment groups than that in control group （P 〈 0.05）. The mRNA expression of Notchl was significantly lower in Hcy-H group than that of other three groups （P 〈 0.05）. The mRNA expression of NOtch 1 was significantly lower in Hcy-M group than that of Hcy-L group and control group （P 〈 0.05）. Conclusion Hcy could inhibit the proliferation of NSCs by down-regulating mRNA expression levels of Notchl and Hesl genes related to Notch signal pathway.