基于c-Kit<sup>+</sup>显微形态标记及流式分选分离心脏Telocytes

doi:10.13523/j.cb.20150107

中国生物工程杂志

2015, Vol. 35

Issue (1): 46-53 DOI: 10.13523/j.cb.20150107

技术与方法

基于c-Kit⁺显微形态标记及流式分选分离心脏Telocytes

肖虎松^1,2,3, 沈晓涛^1,2,3, 郑馨^1,2,3, 李艳梅^1,2,3, 蔡冬青^1,2,3

1. 暨南大学再生医学教育部重点实验室广州 510632;
2. 科技部及广东省国际科技合作基地(暨南大学) 广州 510632;
3. 暨南大学发育与再生生物学系广州 510632

c-Kit⁺ Combining with DiI Micro-label to Sort the Cardiac Telocytes

XIAO Hu-song^1,2,3, SHEN Xiao-tao^1,2,3, ZHENG Xin^1,2,3, LI Yan-mei^1,2,3, CAI Dong-qing^1,2,3

1. Key Laboratory for Regenerative Medicine, Ministry of Education, Jinan University, Guangzhou 510632, China;
2. The International Base of Collaboration for Science & Technology, The Ministry of Science and Technology & Guangdong Province, Jinan University, Guangzhou 510632, China;
3. Department of Developmental and Regenerative Biology, Ji Nan University, Guangzhou 510632, China

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摘要：

目的:建立基于显微形态标记及流式分选分离大鼠心脏Telocytes(CTs)的方法。方法:采用抗体c-Kit免疫磁珠法获得原代心脏Telocytes,显微注射DiI标记具"Telopode"典型形态的细胞,使用流式分离及回收单个DiI⁺细胞;使用免疫荧光技术和RT-PCR方法对经回收的单个细胞来源的细胞进行表型鉴定。结果:显微注射DiI能较好地标记具Telocytes典型形态的细胞,结合流式分选及单细胞回收,能有效回收经标记的DiI⁺细胞,经回收的DiI标记阳性Telocytes的贴壁率为14.9%,增殖率为5.6%,呈克隆样生长率为2.4%,该呈克隆样生长的细胞能通过消化传代。免疫荧光染色证明,该回收Telocytes表达其相对特异性表面标记物c-Kit和CD34,RT-PCR的结果也证明:经回收Telocytes表达其相对特异基因 c-Kit、CD34、Vimentin和PDGFR-β 。结论:研究所建立的方法能有效分离及单细胞回收高纯度的心脏Telocytes,经回收的心脏Telocytes具有增殖及传代能力,且能维持其特异表型。

关键词： 心脏Telocytes; 显微标记; 单细胞分选

Abstract:

Objective: To establish methodology for specific sorting of cardiac telocytes using c-Kit⁺ Combining with DiI Micro-label and flow cytometry. Methods: The cardiac telocytes were isolated via a c-Kit antibody conjugated magnetic bead method. The cells which have unique telocyte morphology, with long "Telopode", were labeled with DiI microinjection. The labeling DiI⁺ cells were sorted and recovered in 96-well plate by single cell flow cytometry technique. The immunofluorescence and RT-PCR were applied to confirm the phenotype of collected cells. Results: The cardiac telocytes (c-Kit⁺) which have unique telocyte morphology were able to be labeled by DiI microinjection. The DiI labeled cells were able to be sorted and recovered in single cell level. The adherent cells of collected DiI⁺ cells were 14.9% under culture. The 5.6% of cultured DiI⁺ cells was able to proliferate and the 2.4% of cultured DiI⁺ cells was able to form cell-clone. In addition, the formed cell-clone was able to subculture. The immunofluorescence staining confirmed that the recovered DiI⁺ cells were c-Kit-and CD34-positive. RT-PCR results also showed that the recovered DiI⁺ cells were positive for c-Kit, CD34, Vimentin and PDGFR-β, which are the markers for telocytes.Conclusion: The methodology which was established in present study was able to isolate and sort cardiac telocytes in single cell level. The sorted cardiac telocytes were able to proliferate and maintain the unique phenotype.

Key words: Cardiac telocytes Microinjection Single cell sorting

收稿日期: 2014-10-22 出版日期: 2015-01-25

ZTFLH:

Q813

基金资助:

科技部港澳台合作专项(2012DFH30060),国家自然科学基金(81170324,81470433),广东省教育厅国际合作平台项目(2012gjhz0003),广东省自然科学基金重点项目(S2012020010895),广东省科技厅国际合作平台项目(2013B051000062)资助项目

通讯作者: 蔡冬青 E-mail: tdongbme@jnu.edu.cn

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引用本文:

肖虎松, 沈晓涛, 郑馨, 李艳梅, 蔡冬青. 基于c-Kit⁺显微形态标记及流式分选分离心脏Telocytes[J]. 中国生物工程杂志, 2015, 35(1): 46-53.

XIAO Hu-song, SHEN Xiao-tao, ZHENG Xin, LI Yan-mei, CAI Dong-qing. c-Kit⁺ Combining with DiI Micro-label to Sort the Cardiac Telocytes. China Biotechnology, 2015, 35(1): 46-53.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150107 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I1/46

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