上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

GFP+Luc双报告基因标记脂肪干细胞裸鼠体内示踪研究

戴 淼1,2，吴氢凯2，许培荣2，候 旻2，冯 洁2   

  1. 1.苏州大学医学院研究生部， 苏州 215123； 2.上海交通大学附属第六人民医院妇产科， 上海 200233
  • 出版日期:2014-08-28 发布日期:2014-09-02
  • 通讯作者: 吴氢凯, 电子信箱: angelh2@163.com。
  • 作者简介:戴 淼(1986—), 女， 硕士生; 电子信箱: hnxxmiao@163.com。
  • 基金资助:

    上海市科委产学研医合作项目（13DZ1941404）

In vivo tracking of adipose-derived stem cells labeled with green fluorescent protein and luciferase dual reporter gene

DAI Miao1,2, WU Qing-kai2, XU Pei-rong2, HOU Min2, FENG Jie2   

  1. 1.Department of Graduate, School of Medicine, Soochow University, Suzhou 215123, China; 2.Department of Obstetrics and Gynaecology, the Sixth People's Hospital, Shanghai Jiao Tong University, Shanghai 200233, China
  • Online:2014-08-28 Published:2014-09-02
  • Supported by:

    Cooperation Project of Medicine and Industry of Science and Technology Commission of Shanghai Municipality, 13DZ1941404

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摘要：

目的 探索慢病毒载体GFP+Luc双报告基因标记和生物发光成像技术活体内示踪脂肪干细胞的可行性。方法 利用慢病毒载体将绿色荧光蛋白（GFP）和荧光素酶（Luc）双基因标记脂肪干细胞，利用荧光显微镜观察人脂肪干细胞（ADSCs）体外转染率，裸鼠皮下注射脂肪干细胞后1 h，1、2、4、6和8周利用生物发光成像系统连续监测ADSCs在裸鼠活体内的发光强度。离体组织DAPI染色鉴定移植细胞的存在及其存在的组织层次。结果 荧光显微镜下观察慢病毒感染后的人ADSCs的GFP表达率高，流式细胞术测定转染率为87.3%，传至25代的人ADSCs仍稳定表达GFP。慢病毒载体对人ADSCs的生长和增殖没有影响。裸鼠注射ADSCs后活体内生物发光时间可持续8周，ADSCs主要分布在移植部位。离体组织切片鉴定标记的脂肪干细胞存在于移植部位皮下。结论 慢病毒介导GFP+Luc双报告基因标记和生物发光技术可用于监测脂肪干细胞在动物体内的生物行为。

关键词: 绿色荧光蛋白, 荧光素酶, 脂肪干细胞, 生物发光成像技术

Abstract:

Objective To explore the feasibility of in vivo tracking of adipose-derived stem cells (ADSCs) by using lentiviral vectors containing green fluorescent protein (GFP) and luciferase (Luc) dual reporter gene transfection and bioluminescent imaging techniques. Methods The ADSCs were isolated from human adipose tissue samples of cosmetic subdermal liposuction. The ADSCs were transfected by lentiviral vectors containing GFP and Luc. The labeling efficiency was evaluated by fluorescence microscope and flow cytometry in vitro. The transfected ADSCs were subcutaneously injected into BALB/C mice, and then a non-invasive bioluminescence imaging procedure was performed 1 h, 1, 2, 4, 6 and 8 weeks after transplantation. The DAPI staining of skin tissues was performed to identify the survival and the location of transplanted ADSCs. Results High GFP expression was observed in transfected ADSCs by lentiviral vector under fluorescence microscope. The transfection efficiency of lentiviral vectors containing GFP and Luc was 87.3%. Transfected ADSCs can express GFP and Luc after 25 successive generations, and the transduction of ADSCs with lentiviral bioluminescence reporter vectors had no effect on cell proliferation. The bioluminescence of ADSCs in vivo can be detectable as long as 8 weeks after transplantion, and the ADSCs mainly survive in the inoculating site of the thigh. Histological analysis of sections showed that the labeled ADSCs located in the subcutaneous layer of the implantation site. Conclusion Using lentiviral vectors containing GFP and Luc dual reporter gene transfection and bioluminescent imaging techniques can monitor ADSCs in vivo.

Key words: green fluorescent protein, luciferase, adipose-derived stem cells, bioluminescence imaging