中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (15): 2773-2777.doi: 10.3969/j.issn.1673-8225.2010.15.027

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

荧光染料CFSE差异浓度标记活细胞体内示踪方法

蒋泽生，高  毅   

  1. 南方医科大学附属珠江医院肝胆二科，南方医科大学再生医学研究所，广东省广州市  515282
  • 出版日期:2010-04-09 发布日期:2010-04-09
  • 通讯作者: 高 毅，博士，教授，南方医科大学附属珠江医院肝胆二科，南方医科大学再生医学研究所，广东省广州市 515282 gaoyi6146@163.com
  • 作者简介:蒋泽生☆，男，1972年生，河南省潢川县人，汉族，1995年毕业于解放军第一军医大学，博士，主治医师，主要从事移植免疫方面的研究。
  • 基金资助:

    国家自然科学基金资助项目(30972825)。课题名称：供体抗原特异性体内免疫状态体内定量检测的基础研究。

A tracing method for mouse spleen lymphocytes using difference concentrations of CFSE in vivo

Jiang Ze-sheng, Gao Yi   

  1. Second Department of Hepatology, Zhujiang Hospital, Southern Medical University, Institute of Regeneration Medicine, Southern Medical Vnirersity, Guangzhou  510282, Guangdong Province, China
  • Online:2010-04-09 Published:2010-04-09
  • Contact: Gao Yi, Doctor, Professor, Second Department of Hepatology, Zhujiang Hospital, Southern Medical University, Institute of Regeneration Medicine, Southern Medical Vnirersity, Guangzhou 510282, Guangdong Province, China zsjiang@hotmail.com
  • About author:Jiang Ze-sheng☆, Doctor, Attending physician, Second Department of Hepatology, Zhujiang Hospital, Southern Medical University, Institute of Regeneration Medicine, Southern Medical Vnirersity, Guangzhou 510282, Guangdong Province, China
  • Supported by:

    the National Natural Science Foundation of China, No. 30972825*

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1340

被引次数

9

摘要：

背景：利用荧光染料标记活细胞体内示踪的方法较多，但是应用荧光染料浓度差异实现不同细胞体内示踪的方法目前仍不成熟。
目的：观察不同浓度荧光活性染料CFSE标记的小鼠脾细胞体内示踪情况，建立稳定的单染料差异浓度标记活细胞的体内示踪方法。
方法：取C57BL/6J及BALB/c小鼠脾脏制备脾细胞单细胞悬液，分别用不同浓度CFSE染色后制备1∶1混合细胞悬液；锥虫蓝染色鉴定细胞活性；将染色的混合细胞输注BALB/c小鼠后不同时间点，流式细胞仪检测小鼠外周血两种荧光细胞的比例。
结果与结论：CFSE标记的小鼠脾细胞活力良好，荧光显微镜下见全部细胞标记后均发绿色荧光，形态正常，CFSE标记阳性率为95%。CFSE染色时，在浓度相差20倍时流式直方图才显示为两个完全独立的峰，即CFSE低浓度用0.3 μmol/L，高浓度用6 μmol/L。异基因的C57BL/6J脾细胞在输注1 d后就快速消失，而同基因BALB/c脾细胞能够在BALB/c小鼠外周血中长期存在，但是两种细胞荧光强度均未见降低，说明荧光染料CFSE差异浓度标记活细胞进行体内示踪是一种稳定的示踪方法。

关键词: 细胞示踪, 体内, 荧光标记, 差异浓度染色, CFSE

Abstract:

BACKGROUND: Many methods have been used in tracing living cells in vivo, but it remains immature to detect cells using difference concentration of fluorochrome.
OBJECTIVE: To establish an assay method for detecting mouse spleen lymphocytes traced by difference concentration of carboxyfluorescein diacetate succinimidyl ester (CFSE) in vivo.
METHODS: Spleen lymphocytes were isolated from C57BL/6J and BALB/c mice and prepared for cell suspension, followed by mixing with difference concentration of CFSE, and then the cell actively was identified by trypan blue staining. The labeled cells were transferred into BALB/c mice. Flow cytometry were performed at different time points to detect the proportion of positive cells.
RESULTS AND CONCLUSION: The CFSE-labeled spleen lymphocytes were highly expressed green fluoresce with a positive labeling rate of 95%. Column diagram showed 2 independent peaks when the concentration of CFSE had 20-fold difference, namely, compared between 0.3 μmol/L and 6 μmol/L. The heterogenic C57BL/6J splenocytes disappeared at 1 day after transfusion, but the syngeneic BALB/c splenocytes could survive for a long time in the peripheral blood of BALB/c mice. The fluorescence intensity was not decreased in two kinds of splenocytes. The results demonstrated that CFSE-labeling with difference concentration is reliable, stable and convenient for spleen lymphocytes tracing in vivo.

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