口腔医学研究

口腔医学研究 ›› 2020, Vol. 36 ›› Issue (6): 523-527.DOI: 10.13701/j.cnki.kqyxyj.2020.06.006

• 口腔生物学研究 • 上一篇    下一篇

人根尖乳头干细胞在羟基磷灰石和聚多巴胺-羟基磷灰石上的粘附、增殖及成骨分化

何林1, 何淞2, 吴稀2, 杨森1*   

  1. 1.遂宁市中心医院口腔医学中心 四川 遂宁 629000;
    2.西南医科大学附属口腔医院 四川 泸州 646000
  • 收稿日期:2019-09-18 出版日期:2020-07-03 发布日期:2020-07-06
  • 通讯作者: *杨森,E-mail:42831594@qq.com
  • 作者简介:何林(1993~ ),女,四川会理人,硕士,医师,主要从事正畸科临床治疗工作。
  • 基金资助:
    四川省科技重点研发项目(编号:2018FZ0113);中华口腔医学会西部行口腔医学临床科研基金(编号:CSA-W-2016-01);四川省科技支撑计划项目(编号:2014SZ0038);四川省医学科研青年创新课题(编号:Q14054)

Adhesion, Proliferation, and Osteogenic Differentiation of hSCAPs on HAP and PDA-HAP

HE Lin1, HE Song2, WU Xi2, YANG Sen1*   

  1. 1. Department of Stomatology, Suining Center Hospital, Suining 629000, China;
    2. Stomatology Hospital, Southwest Medical University, Luzhou 646000, China
  • Received:2019-09-18 Online:2020-07-03 Published:2020-07-06

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摘要： 目的: 提取、鉴定人根尖乳头干细胞(hSCAPs),接种于羟基磷灰石生物陶瓷(HAP)和聚多巴胺-羟基磷灰石生物陶瓷(PDA-HAP)上,对比两种支架的细胞粘附、增殖及成骨分化能力。方法: (1)改良组织块法提取hSCAPs,流式细胞仪鉴定细胞表面抗原:STRO-1、CD90、CD146、CD45、CD34。(2)hSCAPs 分别接种于HAP组和PDA-HAP组,DAPI和鬼笔环肽分别对细胞核和骨架染色,观察细胞形态,计数行统计分析。(3)hSCAPs 分别接种于HAP组和PDA-HAP组,培养1、3、5、7 d,CCK8测量A值检测细胞增殖情况。(4)hSCAPs 接种于HAP组和PDA-HAP组,培养1、7、14 d,检测ALP活性,Real-time PCR检测ALP、OCN和Runx2表达量。结果: (1)所提hSCAPs表达STRO-1(+)、CD90(+)、CD146(+)、CD45(-)、CD34(-)。(2)PDA-HAP组较HAP组上附着的hSCAPs数目更多(P<0.001),且PDA-HAP上的细胞相较HAP上的面积更大,伸展更充分,可见明显的细胞触角。(3)HAP和PDA-HAP培养hSCAPs1、3、5、7 d毒性均无明显差异(P>0.05)。(4)ALP活性:诱导第1天、14天两组活性差异无统计学意义(P>0.05),但第7天PDA-HAP组明显高于HAP组(P<0.001)。Real-time PCR:ALP的表达情况与ALP活性结果一致;Run x2于1、7、14 d均为PDA-HAP组高于HAP组,差异有统计学意义(P<0.05);OCN第1天两组差异无统计学意义(P>0.05),在第7天、14天PDA-HAP组均较HAP组高(P<0.05)。结论: PDA-HAP较HAP能明显促进hSCAPs粘附、成骨相关特异基因ALP、 Runx2、OCN的表达,促进hSCAPs成骨分化。

关键词: 人根尖乳头干细胞, 聚多巴胺, 羟基磷灰石生物陶瓷, 成骨分化

Abstract: Objective: To compare cell adhesion, proliferation, and osteogenic differentiation of hSCAPs on the HAP and PDA-HAP scaffolds. Methods: hSCAPs were extracted by improved tissue block method and cell surface antigens, i.e. STRO-1, CD90, CD146, CD45, and CD34, were identified by flow cytometry. hSCAPs were inoculated on HAP and PDA-HAP, respectively. DAPI and rhodamine phalloidin were used to stain the nucleus and cytoskeleton respectively to observe cell morphology. The cell proliferation was measured by CCK8 after 1, 3, 5, and 7 days. ALP activity and expression of ALP, OCN, and Runx2 were detected after 1, 7, and 14 days. Results: The hSCAPs expressed STRO-1 (+), CD90 (+), CD146 (+), CD45 (-), and CD34 (-). The number of hSCAPs attached to PDA-HAP was more than that on HAP (P<0.001), and the cell area on PDA-HAP was larger and more fully extended than that on HAP. There was no significant difference in toxicity between HAP and P DA-HAP when cultured hSCAPs for 1, 3, 5, and 7 days (P>0.05). There was no significant difference on the ALP activity between two groups on the 1st and 14th day of induction (P>0.05), however, significant difference on the 7th day (P<0.001). By real-time PCR, it was found that the expression of ALP was the same as that of ALP activity; the expressions of Runx2 in PDA-HAP group were higher than those in HAP group on 1, 7, and 14 days (P<0.05); there was no significant difference on the expression of OCN between two groups on the 1st day (P>0.05), however, significant difference on the 7th and 14th day (P< 0.05). Conclusion: PDA-HAP can significantly promote the expression of ALP, Runx2, and OCN, and promote the osteogenic differentiation of hSCAPs.

Key words: human apical papillary stem cells, polydopamine, hydroxyapatite bioceramics, osteogenic differentiation