中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (10): 1759-1763.doi: 10.3969/j.issn.1673-8225.2011.10.011

• 脂肪干细胞 adipose-derived stem cells • 上一篇    下一篇

稳定表达报告基因脂肪间充质干细胞的培养与分子影像鉴定

范伟伟，王亚斌，张荣庆，李聪叶，李  霜，曹  丰   

  1. 解放军第四军医大学西京医院心血管内科，陕西省西安市  710032
  • 收稿日期:2010-10-15 修回日期:2010-11-16 出版日期:2011-03-05 发布日期:2011-03-05
  • 通讯作者: 曹丰，主任医师，教授，博士生导师，解放军第四军医大学西京医院心血管内科，陕西省西安市 710032 wind8828@gmail.com
  • 作者简介:范伟伟★，男，1982年生，北京市人，汉族，解放军第四军医大学在读硕士，医师，主要从事心血管干细胞及分子影像方面的研究。 fww2142008458@gmail.com
  • 基金资助:

    国家自然科学基金项目(30970845)，课题名称为“microRNA-1对人胚胎干细胞心肌分化调控的分子影像研究”；西京医院助推计划重大项目(XJZT08Z04)，课题名称为“胚胎干细胞心肌定向分化及移植后心肌保护作用的机制研究”。

Culture and molecular imaging identification of adipose derived mesenchymal stem cells with stable reporter gene expression in vitro and in vivo

Fan Wei-wei, Wang Ya-bin, Zhang Rong-qing, Li Cong-ye, Li Shuang, Cao Feng   

  1. Department of Cardiology, Xijing Hospital, Fourth Military Medical University of Chinese PLA, Xi’an  710032, Shaanxi Province, China
  • Received:2010-10-15 Revised:2010-11-16 Online:2011-03-05 Published:2011-03-05
  • Contact: Cao Feng, Chief physician, Professor, Doctoral supervisor, Department of Cardiology, Xijing Hospital, Fourth Military Medical University of Chinese PLA, Xi’an 710032, Shaanxi Province, China wind8828@gmail.com
  • About author:Fan Wei-wei★, Studying for master’s degree, Physician, Department of Cardiology, Xijing Hospital, Fourth Military Medical University of Chinese PLA, Xi’an 710032, Shaanxi Province, China fww2142008458@gmail.com
  • Supported by:

    the National Natural Science Foundation of China, No. 30970845*; the Key Assist Project of Xijing Hospital, No. XJZT08Z04*

PDF

386

被引次数

4

摘要：

背景：研究显示分子影像技术可以在活体细胞和分子水平对干细胞进行定性和定量研究，对干细胞长程监测具有独特优势。
目的：构建稳定表达报告基因的脂肪间充质干细胞，并运用报告基因成像等方法实现其体外和移植后的评价与鉴定。
方法：繁殖与筛选携带β-actin-luc报告基因小鼠后，采用改良胶原酶消化法分离培养其脂肪间充质干细胞，取第3代细胞行表面标志鉴定；将脂肪间充质干细胞(1×106)植入BALB/c裸鼠后肢肌肉内，采用萤火虫荧光素酶(fluc)生物发光成像进行体外和体内移植后脂肪间充质干细胞的示踪和定量评价。
结果与结论：转基因小鼠稳定携带β-actin-luc报告基因，分离培养出的脂肪间充质干细胞高表达CD90及CD44，而CD45、CD34 和CD31呈现低表达，其细胞数与fluc报告基因生物发光信号强度呈显著直线相关(r2=0.96)。细胞活体肌肉移植24 h后存活，并显示较强的生物发光信号，在底物荧光素腹腔注射后0~42 min内先迅速增强后平缓减弱，21 min时最强，达(6.92×106±4.11×105) Photons?s-1?cm-2?sr-1。提示由携带β-actin-luc报告基因小鼠脂肪组织分离的脂肪间充质干细胞高表达间充质干细胞表面标志，可在体外和肌肉组织移植后稳定表达该报告基因并实现细胞的分子影像示踪与定量。

关键词: 间充质干细胞, 脂肪间充质干细胞, 报告基因, 分子影像, 干细胞

Abstract:

BACKGROUND: Previous studies show that molecular imaging can monitor the stem cell qualitatively and quantitatively at cellular and molecular levels in vivo and hold promise in long-time evaluation of the stem cell.
OBJECTIVE: To construct adipose derived mesenchymal stem cells (ADMSCs) with stable reporter genes expression and identify it by reporter gene imaging in vitro and in vivo.
METHODS: β-actin-luc transgenic mice were selected firstly.Then ADMSCs were cultured from β-actin-luc mice by modified collagenase method. ADMSCs at passage 3 were identified by flow cytometry. ADMSCs (1×106) were implanted into the muscles of hindlimb of BALB/c nude mice, then tracked and quantified by firefly luciferase (fluc) bioluminescence imaging (BLI) in vitro and in vivo.
RESULTS AND CONCLUSION: The transgenic mice stably carried β-actin-luc reporter gene and the ADMSCs were positive for CD90 and CD44, while negative for CD45, CD34 and CD31. ADMSCs consistently expressed the fluc and a robust correlation existed between different cell numbers and fluc average radiance (r2=0.96). After 24-hours engrafted ADMSCs survived and displayed strong BLI signal, which increased rapidly and then decreased smoothly during 0-42 minutes after peritoneal injection of D-luciferin, the peak being (6.92×106±4.11×105) Photons?s-1?cm-2?sr-1 at 21 minutes. Results indicated that ADMSCs of β-actin-luc transgenic mice could highly express the markers of MSCs, stably carry the reporter gene and facilitate the tracking and quantifying by BLI in vitro and in vivo.

中图分类号: