中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (41): 7631-7636.doi: 10.3969/j.issn.2095-4344.2012.41.007

• 脐带脐血干细胞 umbilical cord blood stem cells • 上一篇    下一篇

CM-Dil及DAPI联合标记示踪人脐血单核细胞细胞膜及细胞核的可行性

何金英1，杨丽敏2，马玉珍1，孙文芳1，岑 尧1，姚星宇3   

  1. 1内蒙古自治区人民医院妇产科， 内蒙古自治区呼和浩特市 010050
    2内蒙古医学院微生物与免疫教研室，内蒙古自治区呼和浩特市 010059
    3满洲里南区医院内科，内蒙古自治区满洲里市 021400
  • 收稿日期:2012-08-01 修回日期:2012-08-17 出版日期:2012-10-07 发布日期:2012-10-07
  • 通讯作者: 姚星宇，副主任医师，满洲里南区医院内科，内蒙古自治区满洲里市 021400 2412602429@qq.com
  • 作者简介:何金英★，女，1972年生，鄂伦春族，内蒙古呼伦贝尔市人，2005年内蒙古医学院毕业，硕士，副主任医师。 1104501492@qq.com

CM-Dil combined with DAPI label cellular membrane and nucleus of human umbilical cord blood mononuclear cells

He Jin-ying1, Yang Li-min2, Ma Yu-zhen1, Sun Wen-fang1, Cen Yao1, Yao Xing-yu3   

  1. 1Department of Obstetrics and Gynecology, the People's Hospital of the Inner Mongolia Autonomous Region, Hohhot 010050, Inner Mongolia Autonomous Region, China 2Laboratory of Microbiology and Immunology, Inner Mongolia Medical College, Hohhot 010059, Inner Mongolia Autonomous Region, China
    3Deapartment of Internal Medicine, Manzhouli Southern District Hospital, Manzhouli 021400, Inner Mongolia Autonomous Region, China
  • Received:2012-08-01 Revised:2012-08-17 Online:2012-10-07 Published:2012-10-07
  • Contact: Yao Xing-yu, Associate chief physician, Department of Internal Medicine, Manzhouli Southern District Hospital, Manzhouli 021400, Inner Mongolia Autonomous Region, China 2412602429@qq.com
  • About author:He Jin-ying★, Master, Associate chief physician, Department of Obstetrics and Gynecology, the People's Hospital of the Inner Mongolia Autonomous Region, Hohhot 010050, Inner Mongolia Autonomous Region, China 1104501492@qq.com

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1127

被引次数

4

摘要：

背景：氯甲基- 1，1 十八烷基-3，3，3’，3’-四甲基-吲哚-羧花青-高氯酸盐(chlormethylbenzamido-1， 1-dioctadecyl-3，3，3’，3’-tetramethylin-docarbocyamine，CM-DiI)和4’，6-联脒-2-苯基吲哚二盐酸盐 (4’，6-diamidino-2-phenylindole，DAPI)是常用的活细胞示踪剂，分别用来标记细胞膜和细胞核。
目的：观察应用细胞膜及细胞核标记物CM-DiI及DAPI联合示踪人脐血单核细胞的可行性及双标后人脐血单核细胞体外培养过程中细胞形态及活性的变化。
方法：将新鲜分离的人脐血单核细胞用示踪剂CM-DiI及DAPI进行双标记，体外培养双标后的人脐血单核细胞，倒置相差显微镜下观察细胞形态变化，锥虫蓝染色检测不同时间细胞活性，同时倒置荧光显微镜观察不同时间经CM-DiI和DAPI双标的人脐血单核细胞荧光染色阳性数。
结果与结论：人脐血单核细胞在CM-DiI和DAPI联合标记15 min后，荧光显微镜下可见标记细胞的细胞膜、细胞核分别在不同波长下分别呈现红色和蓝色的荧光。CM-DiI/DAPI双标后的人脐血单核细胞体外培养1，3，7，14，21 d，CM-DiI和DAPI双染的阳性细胞数各时间点比较差异无显著性意义。锥虫蓝染色观察人脐血单核细胞存活率为95.6%-98.8%。双标后的人脐血单核细胞体外培养过程中细胞形态变化与未经示踪标记的脐血单核细胞相比差异不明显，仍保持了良好的生长状态、贴壁能力和细胞增殖能力。由此证实，CM-DiI和DAPI可有效标记人脐血单核细胞，两种示踪剂对活细胞无毒不良反应，荧光衰减较慢，适用于干细胞标记及示踪。

关键词: 人脐血单核细胞, 荧光染料, CM-DiI, DAPI, 示踪, 形态, 活性, 脐血, 脐血干细胞, 组织工程

Abstract:

BACKGROUND: Chlormethylbenzamido-1, 1-dioctadecyl-3, 3, 3’, 3’-tetramethylin-docarbocyamine (CM-DiI) and 4',6-diamidino-2-phenylindole (DAPI) are two kinds of tracers which are commonly used for labeling cellular membrane and nucleus respectively.
OBJECTIVE: To investigate the feasibility of CM-DiI combined with DAPI labeling human umbilical cord blood mononuclear cells and to observe the changes in morphology and activities of the cells cultured in vitro after double labeling.
METHODS: The freshly isolated mononuclear cells derived from human umbilical cord blood cells were double labeled with CM-DiI and DAPI, and the double labeled human umbilical cord blood cells were cultured in vitro, the changes of the morphology was observed under inverted phase contrast microscope, the activity of the cells at different times was detected though Trypan blue staining, at the same time, human umbilical cord blood cells double labeled by CM-DiI and DAPI were counted under the inverted fluorescence microscope at different times.
RESULTS AND CONCLUSION: The membrane and nucleus of human umbilical cord blood cells present red and blue fluorescence respectively under different wavelengths with fluorescence microscope after double labeling by CM-DiI and DAPI for 15 minutes. The CM-DiI/DAPI double labeled human umbilical cord blood cells were in vitro cultured for 1, 3, 7, 14 and 21 days, and there was no significant difference in the number of positive cells double labeled by CM-DiI and DAPI at different time points under fluorescence microscope. The survival rates were 95.6%-98.8%. There was no significant difference in cell morphology between the in vitro cultured double labeled cells and the cells without labeling, and the growth state, adherent ability and cell proliferation ability of the double labeled cells were not changed. Human umbilical cord blood mononuclear cells can be effectively labeled by CM-DiI and DAPI at the same time. Both of the tracers have slower fluorescence decay and non-toxic adverse reactions on the living cells and are suitable for labeling the stem cells.

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