上海口腔医学

上海口腔医学 ›› 2018, Vol. 27 ›› Issue (2): 123-128.doi: 10.19439/j.sjos.2018.02.003

• 论著 • 上一篇    下一篇

根尖牙乳头干细胞细胞膜片构建及其成骨/成牙本质分化性能研究

马学娟1,2, 刘雪梅1,3, 毕静1, 刘尧1,3, 陈旭1,3   

  1. 1.中国医科大学口腔医学院 儿童口腔科,辽宁 沈阳 110002;
    2.保定市第二医院 口腔科,河北 保定 071000;
    3.辽宁省口腔疾病重点实验室,辽宁 沈阳 110002
  • 收稿日期:2017-08-24 修回日期:2017-10-12 出版日期:2018-04-25 发布日期:2018-05-14
  • 通讯作者: 陈旭,E-mail: chenxu_cmu@sina.com
  • 作者简介:马学娟（1989-）,女,在读硕士研究生,E-mail: mxj1096112842@163.com
  • 基金资助:
    国家自然科学基金（81771059）; 沈阳市科学技术计划项目（F16-205-1-25）

Construction of cell sheets of stem cells from apical papilla and characterization of their biological properties

MA Xue-juan1,2, LIU Xue-mei1,3, BI Jing1, LIU Yao1,3, CHEN Xu1,3   

  1. 1.Department of Pediatric Dentistry, School of Stomatology, China Medical University. Shenyang 110002, Liaoning Province;
    2.Department of Stomatology, Second Hospital of Baoding. Baoding 071000, Hebei Province;
    3.Liaoning Provincial Key Laboratory of Oral Disease. Shenyang 110002, Liaoning Province, China
  • Received:2017-08-24 Revised:2017-10-12 Online:2018-04-25 Published:2018-05-14

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摘要： 目的:构建根尖牙乳头干细胞（stem cells from apical papilla, SCAP）细胞膜片,并对其组织学形态和成骨/成牙本质分化性能进行研究,为SCAP细胞膜片应用于年轻恒牙牙髓再生提供实验基础。方法:分离年轻恒牙第三磨牙牙乳头,进行SCAP原代培养。采用流式细胞术检测SCAP表面标志物,茜素红S染色检测其成骨/成牙本质分化。取第3代SCAP,膜片诱导液连续培养14 d,形成细胞膜片。对SCAP细胞膜片进行H-E染色,组织学观察;流式细胞术检测SCAP细胞膜片的细胞周期变化;实时定量PCR（qRT-PCR）检测细胞膜片中Runx2、ALP、Col-I基因表达;Western印迹法检测细胞膜片Runx2、ALP蛋白表达水平。采用SPSS17.0软件包对实验结果进行统计学分析,两样本细胞周期检测和qRT-PCR基因检测均数的比较采用t检验。结果:组织学H-E染色显示,SCAP细胞膜片由5~6层细胞组成,细胞量大,细胞之间排列紧密,且富含细胞外基质。SCAP细胞膜片细胞周期G2+S期所占比例较低,而G1期比例较高,提示SCAP细胞膜片增殖性能受到显著抑制（P<0.05）。同时,qRT-PCR和Western印迹法结果显示,与SCAP相比,SCAP细胞膜片成骨/成牙本质分化能力显著升高（P<0.05）。结论:SCAP细胞膜片富含细胞外基质,且成骨/成牙本质分化能力高于单纯SCAP,SCAP细胞膜片应用于牙髓再生更具有优势。

关键词: 根尖牙乳头干细胞, 细胞膜片, 牙髓再生

Abstract: PURPOSE: Cell sheets of stem cells from apical papilla (SCAP) were constructed. Their histological and biological properties were studied to provide experimental basis for its application in dental pulp regeneration in immature permanent teeth. METHODS: SCAP were isolated and cultured from immature permanent third molars. Flow cytometry was used to evaluate surface marker expression of SCAP. Alizarin red S staining was used to test osteo/odontogenic differentiation capacity of SCAP. SCAP (at P3) were cultured in cell sheets medium for 14 days. Hematoxylin-eosin(H-E) staining was used for histological observation; flow cytometry analysis was used to test cell cycle of SCAP cell sheets; Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression levels of runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and collagen I (Col-I). Western blot was used to detect the protein expression levels of Runx2 and ALP. SPSS 17.00 software package was used for statistical analysis. RESULTS: H-E staining showed that SCAP cell sheets contained multiple layers of cells (5 to 6 layers) with high cell density, retained tight junctions and secreted rich extracellular matrix. The cell cycle of SCAP cell sheets showed that G2+S phase was lower, while G1 phase was higher than SCAP, which indicated the proliferation rate of SCAP cell sheets was down-regulated (P<0.05). Moreover, compared with SCAP, osteo/odontogenic differentiation capacity of SCAP cell sheets was significantly increased (P<0.05). CONCLUSIONS: SCAP cell sheets secret rich extracellular matrix, and osteo/odontogenic differentiation capacity in SCAP cell sheets is higher than SCAP. SCAP cell sheets may possess more potentials in dental pulp regeneration.

Key words: Stem cells from apical papilla, Cell sheets, Dental pulp regeneration

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