人牙龈干细胞的分离鉴定及基质细胞衍生因子-1对其趋化效应的研究

doi:10.7518/hxkq.2015.03.005

华西口腔医学杂志

人牙龈干细胞的分离鉴定及基质细胞衍生因子-1对其趋化效应的研究

杜令倩1 杨丕山2 葛少华2

1.山东大学第二医院口腔科，济南 250033；2.山东大学口腔医院牙周病科山东省口腔生物医学重点实验室，济南 250012

出版日期:2015-06-01 发布日期:2015-06-01
通讯作者: 葛少华，副教授，博士，E-mail：shaohuage@sdu.edu.cn
作者简介:杜令倩，住院医师，硕士，E-mail：281790589@qq.com
基金资助:
国家自然科学基金资助项目（81371157，81100756）；山东省科技攻关计划基金资助项目（2014GSF118075）

Culturing and characterization of human gingival mesenchymal stem cells and their chemotactic responses to stromal cell-derived factor-1

Du Lingqian1, Yang Pishan2, Ge Shaohua2.

1. Dept. of Stomatology, The Second Hospital of Shandong University, Jinan 250033, China; 2. Dept. of Periodontology, School of Stomatology, Shandong University, Shandong Provincial Key Laboratory of Oral Biomedicine, Jinan 250012, China

Online:2015-06-01 Published:2015-06-01

摘要/Abstract

摘要：

目的研究基质细胞衍生因子-1（SDF-1）受体CXCR4在人牙龈干细胞（GMSCs）上的表达及SDF-1对人GMSCs的趋化效应。方法通过有限稀释法分离并培养人GMSCs，检测其表面干细胞标志物的表达情况，测试其克隆形成率及多向分化能力，利用免疫荧光染色法检测人GMSCs上SDF-1受体CXCR4的表达，用Transwell细胞培养室检测不同质量浓度SDF-1对人GMSCs的趋化反应，光镜下计数迁移至滤膜下侧面的不同视野的细胞数。结果人GMSCs具有较高的自我更新能力，在体外呈克隆状生长，表达间充质干细胞表面标志物CD44、CD73、CD90、CD105和CD166，而造血干细胞表面标志物CD14、CD34和CD45的表达为阴性。体外诱导培养的人GMSCs能够向成骨细胞及成脂细胞分化，其克隆形成率为21.4%±2.8%。免疫荧光染色显示，人GMSCs表达SDF-1受体CXCR4。SDF-1的质量浓度为100、200 ng·mL^-1时，Transwell细胞培养室中迁移的细胞数目（每高倍视野分别为189.3±4.4和164.6±4.9）显著多于空白对照组（每高倍视野47.8±2.5）（P<0.01）；使用CXCR4中和抗体处理后，人GMSCs的迁移效应明显受到抑制（每高倍视野降低为29.0±2.4，P<0.01）。结论人GMSCs表达趋化因子SDF-1受体CXCR4，SDF-1对人GMSCs有趋化效应，这种趋化效应可能是通过其特异性受体CXCR4介导的。

关键词: 基质细胞衍生因子-1, 牙龈干细胞, 趋化作用

Abstract:

Objective To investigate the expression of chemokine stromal cell-derived factor-1 (SDF-1) receptor CXCR4 in human gingival mesenchymal stem cells (GMSCs) and the migration potential of GMSCs stimulated with SDF-1. Methods Human GMSCs were isolated by single-cell cloning method. Their cell surface markers were characterized by flow cytometry, and the rate of colony formation was evaluated. Differentiation assay was used to detect the differentiation potential of GMSCs. The expression of chemokine SDF-1 receptor CXCR4 in GMSCs was detected by immunocytochemical staining. The chemotactic effect of SDF-1 on GMSCs was detected using a 24-multiwell Transwell cell culture chamber. The number of net migrated cells was counted in different microscope fields. Results Human GMSCs possessed high self-renewal potential and formed single-cell colonies cultured in vitro. GMSCs expressed mesenchymal stem cells-associated markers CD44, CD73, CD90, CD105, and CD166, and the expression of hemopoietic stem cell surface markers CD14, CD34, and CD45 was negative. GMSCs differentiated into osteoblasts and adipocytes under defined culture conditions. The colony forming unit-fibroblastic for GMSCs was 21.4%±2.8%. Immunocytochemical staining demonstrated that GMSCs expressed chemokine SDF-1 receptor CXCR4. The number of GMSCs migrating at concentrations of 100 ng·mL^-1and 200 ng·mL^-1of SDF-1 in the Transwell cell culture chamber was significantly higher than that of the negative control (189.3±4.4, 164.6±4.9 cells/field vs. 47.8±2.5 cells/field, P<0.01). Treatment with the CXCR4 neutralizing antibody, an antagonist for CXCR4, significantly reduced the migratory effect compared with the negative controls (29.0±2.4 cells/field vs. 47.8±2.5 cells/field, P<0.01). Conclusion Human GMSCs express chemokine SDF-1 receptor CXCR4. SDF-1 may participate in regulating chemotaxis of human GMSCs. Results suggest that the migration induced by SDF-1 is mediated by CXCR4.

Key words: stromal cell-derived factor-1, gingival mesenchymal stem cells, chemotaxis

杜令倩杨丕山葛少华. 人牙龈干细胞的分离鉴定及基质细胞衍生因子-1对其趋化效应的研究[J]. 华西口腔医学杂志, doi: 10.7518/hxkq.2015.03.005.

Du Lingqian, Yang Pishan, Ge Shaohua.. Culturing and characterization of human gingival mesenchymal stem cells and their chemotactic responses to stromal cell-derived factor-1[J]. West China Journal of Stomatology, doi: 10.7518/hxkq.2015.03.005.

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人牙龈干细胞的分离鉴定及基质细胞衍生因子-1对其趋化效应的研究

Culturing and characterization of human gingival mesenchymal stem cells and their chemotactic responses to stromal cell-derived factor-1

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