天津医药

• 实验研究 •    

携带标记基因的慢病毒载体转染人脐带华通胶间充质干细胞的实验研究

王力1,张宁坤2,高连如2,朱智明2,徐小红2   

  1. 1. 第二军医大学海军临床医学院,中国人民解放军海军总医院心脏中心
    2. 中国人民解放军海军总医院心脏中心
  • 收稿日期:2013-01-24 修回日期:2013-05-03 出版日期:2013-10-15 发布日期:2013-10-15
  • 通讯作者: 王力

Transfection of Lentivirus Recombined with Marker Gene into Human Umbilical Cord Wharton’s Jelly-Derived Mesenchymal Stem Cells

WANG Li 1,ZHANG Ning kun2,GAO Lian ru2,ZHU Zhi ming2,XU Xiao hong2   

  1. 1. Clinical College of Navy Medicine, Second Military Medical University Department of Cardiology, Navy General Hospital
    2. Department of Cardiology, Navy General Hospital
  • Received:2013-01-24 Revised:2013-05-03 Published:2013-10-15 Online:2013-10-15
  • Contact: WANG Li

PDF (PC)

2364

摘要：

【摘要】 目的  探讨含有增强型绿色荧光蛋白标记基因的慢病毒（Lentivirus-EGFP）体外转染人脐带华通胶间充质干细胞(HUWMSCs)的最佳方案及其对细胞增殖活性的影响。 方法  设计A、B、C、D共4种转染方案，分别以1、10、100的感染复数（MOI）转染体外培养的HUWMSCs，荧光显微镜及流式细胞仪检测各组荧光表达强度及转染效率，MTT法检测细胞增殖倍数。  结果  转染4d后所有实验组转染率在10.6%～87.3%，并与MOI值存在量效关系，聚凝胺（5mg/L）可显著增加病毒转染效率（P＜0.05）。MTT实验结果显示，与对照组比较，不同转染方案细胞增殖存在显著差异（P＜0.001）。A方案MOI=10组及B方案MOI=10组细胞增殖情况较其他组好。 结论  B方案MOI=10实验组为最佳转染方案。Lentivirus-EGFP能高效转染HUWMSCs且在2周内稳定表达转染基因，是一种安全、有效的基因转移载体。

关键词: 间质干细胞, 流式细胞术, 慢病毒属, 遗传载体, 基因疗法, 转染, 细胞增殖

Abstract:

[Abstract]   Objective   To investigate the optimal condition of lentivirus，which was recombined with marker gene of
enhanced green fluorescent protein (Lentivirus-EGFP) transfect human umbilical cord wharton’s jelly-derived mesenchymal stem cells (HUWMSCs) and the effect of transfection on the proliferation in HUWMSCs.  Methods   HUWMSCs were transfected with EGFP by lentivirus vector in vitro via different multiplicity of transfection (MOI) in four different transfection methods (A, B, C and D). The fluorescence expression and the transfection efficiency in different methods were analyzed by both fluorescent microscope and flow cytometry. The proliferation rates of infected HUWMSCs was evaluated by MTT method.  Results   The transfection efficiency was10.6%-87.3% after4days in all experimental groups, which showed the dose-effect relationship with MOI. Polybrene (5mg/L) could significantly increase the transfection efficiency (P＜0.05). Results of MTT assay showed that there were significant differences in the proliferation rates of infected HUWMSCs between different transfection methods (P<0.001). There was better cell proliferation in method A (MOI=10) group and method B (MOI=10) group than that of other groups.   Conclusion   Method B (MOI=10) is the optimal transfection method in this experiment. HUWMSCs could be transfected by lentivirus-EGFP with high efficiency and could stably express transfected gene within2weeks, which is a safe and effective gene transfer vector.

Key words: mesenchymal stem cells, flow cytometry, lentivirus, genetic vectors, gene therapy, transfection, cell proliferation