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山东大学学报（医学版） ›› 2015, Vol. 53 ›› Issue (1): 34-40.doi: 10.6040/j.issn.1671-7554.0.2014.552

• 基础医学 • 上一篇    下一篇

地黄多糖对过表达Notch1(NICD)大鼠骨髓间充质干细胞诱导分化及增殖的影响

付海燕1, 胡占升1, 杜红阳2, 李潮3, 包翠芬4   

  1. 1. 辽宁医学院附属第一医院ICU, 辽宁 锦州 121001;
    2. 辽宁医学院附属第一医院皮肤科, 辽宁 锦州 121001;
    3. 辽宁医学院附属第一医院病理科, 辽宁 锦州 121001;
    4. 辽宁医学院解剖学教研室, 辽宁 锦州 121001
  • 收稿日期:2014-08-26 修回日期:2014-12-09 发布日期:2015-01-10
  • 通讯作者: 杜红阳。E-mail:4197123@163.com E-mail:4197123@163.com
  • 基金资助:
    辽宁省自然科学基金(2013022059);辽宁省教育厅科学技术研究项目基金(L2010275);辽宁医学院青年科技启动基金(Y2011Z010)

Effect of rehmannia glutinosa polysaccharide on the differentiation and proliferation of over-expressed Notch1(NICD) gene in rat bone marrow mesenchymal stem cells in vitro

FU Haiyan1, HU Zhansheng1, DU Hongyang2, LI Chao3, BAO Cuifen4   

  1. 1. Department of ICU, First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001, Liaoning, China;
    2. Department of Dermatology, First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001, Liaoning, China;
    3. Department of Pathology, First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001, Liaoning, China;
    4. Department of Anatomy, Liaoning Medical University, Jinzhou 121001, Liaoning, China
  • Received:2014-08-26 Revised:2014-12-09 Published:2015-01-10

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摘要： 目的 探讨地黄多糖对过表达Notch1(NICD)大鼠骨髓间充质干细胞(BMSCs)诱导分化及增殖的影响。方法 以P3～P5代大鼠BMSCs为研究对象,将细胞随机分为正常对照组、空载体组和转染组。脂质体法转染Notch1(NICD)过表达真核载体,转染后以地黄多糖(0.2 mg/mL)进行诱导分化,转染和诱导后行细胞一般状态观察,采用免疫细胞化学法和Western blotting法检测Notch1(NICD)蛋白、神经元特异性烯醇化酶(NSE)和胶质纤维酸性蛋白(GFAP)表达,CCK-8检测细胞存活率。结果 转染后转染组细胞似神经胶质样细胞形态改变,诱导后各组细胞似神经元样细胞形态结构,转染组显著。转染组Notch1和GFAP蛋白表达阳性率显著高于正常对照组和空载体组(P<0.05),而NSE在3组间差异无统计学意义(P>0.05);诱导后转染组NSE阳性率高于正常对照组和空载体组(P<0.05)。转染后与诱导后比较,转染组Notch1表达阳性率降低(P<0.05),且正常对照组与空载体组亦降低(P<0.05);转染组NSE表达阳性率明显升高(P<0.01),且正常对照组与空载体组明显升高(P<0.01);转染组GFAP表达阳性率明显降低(P<0.01)。CCK-8检测显示,地黄多糖诱导后,随时间延长各组细胞存活率略呈下降趋势。 结论 转染高表达Notch1(NICD)大鼠BMSCs具有向神经胶质样细胞分化倾向,地黄多糖显著抑制Notch1蛋白的表达,诱导BMSCs向神经元样细胞分化。

关键词: 神经元特异性烯醇化酶, 转染, 细胞分化, Notch信号通路, 骨髓间充质干细胞, 诱导

Abstract: Objective To explore the effect of rehmannia glutinosa polysaccharide (RGP) on the differentiation and proliferation of over-expressed Notch1(NICD) gene in rat bone marrow mesenchymal stem cells (BMSCs) in vitro. Methods The P3 to P5 of BMSCs were randomly divided into the control group (CON group), empty vector group (VEC group) and transfection group (TRA group). The NICD eukaryotic expression vectors were transfected into BM-SCs which were then induced with RGP (concentration 0.2 mg/mL). The general morphology was observed. Protein expression of NICD, neurone specific enolase (NSE) and glial gibrillary acidic protein (GFAP) were detected with immunocytochemetry assay and Western blotting assay. Cell survival rate was assessed with CCK-8 assay. Results The BMSCs in TRA group were glial-like cells after transfection, and the induced cells were neuron-like, especially in TRA group. Immunofluorescence assay showed that the positive cell percentage of Notch1 and GFAP protein expressions in TRA group were significantly higher than those in CON group and VEC group (P<0.05). However, NSE in the three groups had no statistical difference after transfection (P>0.05). The positive cell percentage of NSE in TRA group was remarkably higher than that in CON group and VEC group after induction (P<0.05). The positive rate of Notch1 expression in TRA, CON and VEC groups decreased (P<0.05). The positive rate of NSE expression in TRA, CON and VEC group markedly increased (P<0.01). The positive rate of GFAP expression in TRA group decreased significantly (P<0.01). The CCK-8 assay showed that cell survival rate decreased with time in each group after RGP induction. Conclusion The transfection of NICD over eukaryotic expression vector may induce rat BMSCs into glial-like cells. RGP inhibits the expression of Notch1 protein significantly and induces BMSCs into neuron like cells.

Key words: Notch signal pathway, Transfection, Cell differentiation, Neurone specific enolase, Induction, Mesenchymal stem cells

中图分类号: 

  • R285.5
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