天津医药

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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

林宁宁1,张绪梅1,秦善春1,罗素慧1,黄国伟2   

  1. 1. 天津医科大学公共卫生学院营养与食品卫生学系
    2. 天津医科大学
  • 收稿日期:2013-03-22 修回日期:2013-06-18 出版日期:2013-09-15 发布日期:2013-09-15
  • 通讯作者: 林宁宁

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

LIN ning ning1,ZHANG Xu mei1,QIN Shan chun1,LUO Su hui1,HUANG Guo wei2   

  1. 1. Department of Nutrition and Food Science, School of Public Health, Tianjin Medical University
    2. Tianjin Medical University
  • Received:2013-03-22 Revised:2013-06-18 Published:2013-09-15 Online:2013-09-15
  • Contact: LIN ning ning

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摘要：

【摘要】 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Hes1mRNA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-tubulin Ⅲ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6d后，形成β-tubulin Ⅲ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Hes1mRNA表达低于对照组(P<0.05)。Hcy-H组Notch1mRNA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Hes1mRNA表达有关

关键词: 半胱氨酸, 干细胞, 神经元, 细胞增殖, 受体, Notch1, Hes1

Abstract:

[Abstract]   Objective   To explore the effects of homocysteine (Hcy) on neural stem cell (NSC) proliferation and the
mRNA expression level of Notch1and Hes1related 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 Ⅲ and GFAP. The proliferation ability of NSCs was detected by MTT. The mRNA expressions of Notch1and Hes1were 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 Ⅲ positive neurons and GFAP positive astrocytes were detected by immunofluorescence staining on the6th 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 Hes1was significantly lower in three Hcy treatment groups than that in control group (P<0.05). The mRNA expression of Notch1was significantly lower in Hcy-H group than that of other three groups (P<0.05). The mRNA expression of Notch1was 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 Notch1and Hes1genes related to Notch signal pathway

Key words: cysteine, neuron, cell proliferation, receptor, Notch1, Hes1