中国组织工程研究 ›› 2015, Vol. 19 ›› Issue (6): 861-867.doi: 10.3969/j.issn.2095-4344.2015.06.008

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

P2X7受体对牙周膜干细胞成骨分化的作用

孙亚男,魏建敏,孙  薇,武俊杰,吴礼安   

  1. 解放军第四军医大学口腔医院儿童口腔科,军事口腔医学国家重点实验室,陕西省西安市 710032
  • 收稿日期:2015-01-10 出版日期:2015-02-05 发布日期:2015-02-05
  • 通讯作者: 吴礼安,博士,副教授,解放军第四军医大学口腔医院,军事口腔医学国家重点实验室,陕西省西安市 710032 并列通讯作者:武俊杰,博士,讲师,解放军第四军医大学口腔医院,军事口腔医学国家重点实验室,陕西省西安市 710032
  • 作者简介:孙亚男,女,1990年生,河北省保定市人,汉族,解放军第四军医大学在读硕士,医师,主要从事儿童口腔疾病的诊断与治疗。
  • 基金资助:

    2011年国家自然科学基金青年科学基金项目(81100750);国家自然科学基金面上项目(81170929);2014年陕西省自然科学基金面上项目(2014JM4114)

Role of P2X7 receptor in osteogenic differentiation of periodontal ligament stem cells

Sun Ya-nan, Wei Jian-min, Sun Wei, Wu Jun-jie, Wu Li-an   

  1. Department of Pediatric Stomatology, Stomatological Hospital of Fourth Military Medical University of PLA, State Key Laboratory of Military Stomatology, Xi’an 710032, Shaanxi Province, China
  • Received:2015-01-10 Online:2015-02-05 Published:2015-02-05
  • Contact: Wu Li-an, M.D., Associate professor, Department of Pediatric Stomatology, Stomatological Hospital of Fourth Military Medical University of PLA, State Key Laboratory of Military Stomatology, Xi’an 710032, Shaanxi Province, China Corresponding author: Wu Jun-jie, M.D., Lecturer, Department of Pediatric Stomatology, Stomatological Hospital of Fourth Military Medical University of PLA, State Key Laboratory of Military Stomatology, Xi’an 710032, Shaanxi Province, China
  • About author:Sun Ya-nan, Studying for master’s degree, Physician, Department of Pediatric Stomatology, Stomatological Hospital of Fourth Military Medical University of PLA, State Key Laboratory of Military Stomatology, Xi’an 710032, Shaanxi Province, China
  • Supported by:

    the National Natural Science Foundation of China for the Youth in 2011, No. 81100750; the National Natural Science Foundation of China, No. 81170929; the Natural Science Foundation of Shaanxi Province in 2014, No. 2014JM4114

摘要:

背景:研究影响牙周膜干细胞成骨分化的相关离子通道。
目的:初步观察P2X7受体在人牙周膜干细胞成骨分化中的作用。
方法:分离培养人牙周膜干细胞,分为4组,分别添加成骨诱导液和100 nmol/L三磷酸腺苷、成骨诱导液、100 nmol/L三磷酸腺苷及普通培养基培养,于成骨诱导7,14 d,利用茜素红染色检测成骨效果,qRT-PCR,Western blot检测OCN、Runx2以及P2X7受体的表达。
结果与结论:茜素红染色显示成骨诱导液+三磷酸腺苷组牙周膜干细胞的成骨效果在7 d时显著好于成骨诱导液组,但在14 d时成骨诱导液组成骨效果显著好于成骨诱导液+三磷酸腺苷组(P < 0.05);在成骨诱导7 d时,qRT-PCR结果显示成骨诱导液+三磷酸腺苷组Runx2,OCN mRNA的表达达到峰值,而14 d时明显降低(P < 0.05)。成骨诱导7 d时,成骨诱导液+三磷酸腺苷组P2X7受体mRNA的表达量明显高于三磷酸腺苷组(P < 0.05),成骨诱导液组和对照组未见P2X7受体mRNA的表达,成骨诱导14 d时,成骨诱导液+三磷酸腺苷组表达量下降,明显低于三磷酸腺苷组,差异有显著性意义(P < 0.05)。Western blot结果显示,成骨诱导7 d时,成骨诱导液+三磷酸腺苷组P2X7受体的表达达到峰值,高于三磷酸腺苷组,而成骨诱导液组表达量低,对照组几乎没有表达;成骨诱导14 d时,成骨诱导液+三磷酸腺苷组P2X7受体的表达比7 d时明显下降,而三磷酸腺苷组P2X7受体的表达比7 d时有所增加。结果表明外源性三磷酸腺苷可在牙周膜干细胞成骨诱导前7 d明显提高成骨效果,但在7 d后,抑制成骨诱导效果,三磷酸腺苷可以激活牙周膜干细胞P2X7受体表达,且P2X7受体的表达与牙周膜干细胞的成骨效果正相关。


中国组织工程研究杂志出版内容重点:干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程


全文链接:

关键词: 干细胞, 诱导, 牙周膜干细胞, 培养, 成骨分化, P2X7受体, RUNX2, OCN, 国家自然科学基金

Abstract:

BACKGROUND: Ion channels related to the osteogenic differentiation of periodontal ligament stem cells have been studied.
OBJECTIVE: To preliminarily investigate the effect of P2X7 receptor on the osteogenic differentiation of human periodontal ligament stem cells.
METHODS: Human periodontal ligament stem cells were isolated and divided into four groups, cultured in
100 nmol/L adenosine triphosphate+osteogenic medium, osteogenic medium, 100 nmol/L adenosine triphosphate, and normal culture medium (control group), respectively. After induction for 7 and 14 days, osteogenic effect was detected by alizarin red staining, and expressions of OCN, Runx2 and P2X7 receptor at mRNA and protein levels were analyzed by qRT-PCR and western blot assay, respectively.
RESULTS AND CONCLUSION: Under alizarin red staining, the osteogenic effect of periodontal ligament stem 
cells was better in the group of adenosine triphosphate+osteogenic medium than the osteogenic medium group at 7 days, but it was better in the osteogenic medium group than the group of adenosine triphosphate+osteogenic medium at 14 days (P < 0.05). Results from qRT-PCR showed that, in the group of adenosine triphosphate+osteogenic medium, the mRNA expression of Runx2 and OCN reached peak at 7 days, but decreased significantly at 14 days (P < 0.05). The expression of P2X7 receptor mRNA in the group of adenosine triphosphate+osteogenic medium was significantly higher than that in the adenosine triphosphate group at 7 days (P < 0.05), but significantly lower than that in the adenosine triphosphate group at 14 days (P < 0.05). There was no expression of P2X7 receptor mRNA in the osteogenic medium group and control group. Western blot assay showed that at 7 days, the expression of P2X7 receptor peaked in the adenosine triphosphate+osteogenic medium group at 7 days, which was significantly higher than that in the adenosine triphosphate group, and there was a low expression of P2X7 receptor in the osteogenic medium group and no expression in the control group; at 14 days, the expression of P2X7 receptor decreased significantly in the adenosine triphosphate+osteogenic medium group but increased in the adenosine triphosphate group. Experimental findings show that exogenous adenosine triphosphate could significantly improve the osteogenic differentiation of periodontal ligament stem cells at 7 days, but after that, inhibit the osteogenic differentiation; exogenous adenosine triphosphate could activate the expression of P2X7 receptor in periodontal ligament stem cells, and P2X7 receptor expression has positive correlation with the osteogenic differentiation of periodontal ligament stem cells.


中国组织工程研究杂志出版内容重点:干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程


全文链接:

Key words: Stem Cells, Periodontal Ligament, Receptors, Purinergic P2X7, Adenosine Triphosphate

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