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Notch1(NICD)过表达真核载体构建及对大鼠BMSCs增殖分化的影响

杜红阳   

  1. 辽宁医学院附属第一医院
  • 收稿日期:2014-05-09 修回日期:2014-05-28 出版日期:2014-09-15 发布日期:2014-09-15
  • 通讯作者: 杜红阳

Construction of Notch1(NICD) eukaryotic expression vector and its influence on the proliferation and differentiation of rat bone marrow mesenchymal stem cells in vitro

Hongyang DU   

  1. Department of Dermatology,Liaoning Medical University, Jinzhou121001,China
  • Received:2014-05-09 Revised:2014-05-28 Published:2014-09-15 Online:2014-09-15
  • Contact: Hongyang DU

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摘要： 目的 构建Notch1(NICD)过表达真核载体，探讨其对大鼠骨髓间充质干细胞（bone mesenehymal stem cells，BMSCs）增殖分化的影响。方法 以大鼠BMSCs为研究对象，构建Notch1(NICD)过表达真核载体，将pEGFP-N1-NICD质粒转染BMSCs，实验分为空白对照CON组、空载体组和转染组，转染48h后观察细胞一般形态，Realtime PCR和Western blotting检测NSE、GFAP 和Notch1基因和蛋白表达，流式检测转染后细胞凋亡和细胞周期情况；MTT检测增殖情况。结果 经DNA测序结果，重组质粒pEGFP-N1-NICD编码序列与设计完全一致，转染48h后转染组和空载体组的BMSCs均可表达绿色荧光。转染组Notch1、GFAP基因相对表达量显著高于空载体组和CON组（P＜0.05），Western blotting检测结果与之类似。转染48h后，转染组活细胞比率及早期凋亡率、晚期凋亡率与CON组和空载体组比较差异均具有统计学意义（P＜0.05）；空载体组与CON组晚期凋亡率比较差异具有统计学意义（P＜0.05），转染组细胞处于G1/G0细胞比例显著高于CON组和空载体组（P＜0.01），而处于S和G2/M期细胞比例显著低于CON组和空载体组（P＜0.01），转染组增殖曲线值随时间逐渐低于CON组和空载体组，到第4d时显著低于CON组和空载体组（P＜0.05）。结论Notch1(NICD)过表达真核载体构建成功，高表达Notch1(NICD)基因可能在一定程度上诱导BMSCs凋亡、抑制其增殖，且表现诱导向神经胶质样细胞分化。

关键词: Notch信号通路, 骨髓间充质干细胞, 基因转染, 诱导

Abstract: [Abstract] Objective To investigate the effect of construct theNotch1(NICD) eukaryotic expression vector on the proliferation and differentiation of rat bone marrow mesenchymal stem cells (BMSCs)in vitro. Methods Rat BMSCs were experimented as the object.NICDeukaryotic expression vector was constructed. pEGFP- N1- NICD expressing plasmids were used to transfect BMSCs. The study included control group (CON group), empty vector group (VEC group) and the trans? fection group (TRA group). After 48-hour transfection, BMSCs were observed for general morphology. The protein expres? sions of NSE, GFAP and Notch1were detected by real-time PCR and Western blotting assay respectively. The apoptosis, cy? cle distribution and cell proliferation were evaluated by flow cytometry and MTT assay.Results The DNA sequencing con? firmed that the pEGFP-N1-NICD recombinant plasmid was successfully constructed, and both VEC group and TRA group expressed green fluorescence after48-hour transfection. The relative expression levels of Notch1and GFAP mRNA and pro? tein were significantly higher in TRA group than those in VEC group and CON group (P<0.05), and there was no significant difference between VEC group and CON group. After48-hour transfection, the ratio of living cells was significantly lower in TRA group than that of CON group and VEC group, and the early apoptotic rate and late apoptotic rate were significantly higher in TRA group than those of CON group and VEC group(P<0.05). The late apoptotic rate was significantly higher in  VEC group than that of CON group. The proportion of G1/G0cells was significantly higher in TRA group than that of CON
group and VEC group, but S and G2/M cells were significantly lower (P <0.05). The value of growth curve was gradually de?
creased in TRA group than that of CON group and VEC group (P＜0.05). Conclusion The high expression ofNICDgene
might induce apoptosis of BMSCs, inhibit the proliferation in part, and induce into glial-like cell differentiation

Key words: Notch signal pathway, mesenchymal stem cells, gene transfection, induction