天津医药

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不同原代培养方法培养人牙髓细胞的研究

沈晴昳   

  1. 上海市口腔病防治院
  • 收稿日期:2010-05-11 修回日期:2010-09-10 出版日期:2011-02-15 发布日期:2011-02-15
  • 通讯作者: 沈晴昳

Study on different methods in primary cultures of human dental pulp cells in vitro

  • Received:2010-05-11 Revised:2010-09-10 Published:2011-02-15 Online:2011-02-15

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摘要： 目的：比较并建立一种操作简单方便的人牙髓细胞原代培养方法。方法：选择因正畸拔除的完整无龋双尖牙，无菌条件下取出牙髓，分别采用组织块法、酶消化法和改良组织块法进行人牙髓细胞的体外原代培养，比较观察三种不同培养方法的成活率、细胞形态和数量，测定生长曲线并进行细胞来源鉴定。结果：三种方法均可从人牙髓组织分离培养获得外胚间充质来源的牙髓细胞，呈成纤维细胞样，抗波形丝蛋白染色阳性，抗角蛋白染色阴性。组织块法耗时较长，培养成功率26.7%；酶消化法培养成活率较低,且细胞数量较少,培养成功率仅为13.3%；在改良组织块法中未发现有漂浮的组织块，培养成活率较高为80%。结论：改良组织块法解决了传统组织块法中组织块难以贴壁的问题，简化了操作步骤并具有较高的成功率,是一种科学可行的人牙髓细胞原代培养方法。

关键词: 人牙髓细胞, 细胞培养, 原代培养, 组织块法

Abstract: Objective　To compare the effects of different methods and explore a simple primary culture technique of human dental pulp cell in vitro. Methods: Pulp tissue was separated from healthy young human teeth extracted for orthodontic purpose. The pulp explants were cultured by the tissue explants method, tissue-explants collagenase /dispase digestion method and modified tissue culture techniques respectively. The cell culture efficiency and cell morphology were observed and evaluated by phase-contract microscopy. Immunocytochemical staining method was used to characterize the dental pulp cell lineage. Results: The cultured cells appeared to be fibroblast-like cells．Anti-vimentin was positive and anti?cytokeratin was negative by immunohistochemical stain. The primary dental pulp cells cultured by the tissue explants method could reach confluence after 4 weeks and the culture successfulness was 26.7%. The culture successfulness of dental pulp cells obtained by collagenase /dispase digestion method was only 13.3%. Dental pulp cells can be obtained by the modified tissue culture without floating tissue fragments and the success rate could reach to 80%. Conclusion: Modified tissue culture techniques solved the difficulty of adherence of tissue fragments, simplified the procedure, and kept a high success rate. The modified tissue culture established in this study is an effective method for the primary culture of human dental pulp cells in vitro.

Key words: Human dental pulp cells, Cell culture, Primary culture, Tissue explants