淫羊藿次苷对犬颌骨间充质干细胞增殖与成骨分化的影响

doi:10.19438/j.cjoms.2019.04.004

中国口腔颌面外科杂志 ›› 2019, Vol. 17 ›› Issue (4): 304-310.doi: 10.19438/j.cjoms.2019.04.004

淫羊藿次苷对犬颌骨间充质干细胞增殖与成骨分化的影响

张意闻¹, 罗远^1,2, 郭华艳³, 张迎娣³, 缪国俊³, 黄远亮³

1.同济大学口腔医学院口腔颌面外科教研室,上海牙组织与再建工程技术研究中心,上海 200072;
2.上海市口腔病防治院口腔颌面外科,上海 200031;
3.同济大学附属东方医院口腔科,上海 200120

收稿日期:2018-12-03 修回日期:2019-01-21 出版日期:2019-07-20 发布日期:2019-08-12
通讯作者: 黄远亮,E-mail: ylhuang0115@163.com
作者简介:张意闻（1993-）,女,硕士研究生,E-mail: zhangyiwen0129@163.com
基金资助:
国家自然科学基金（81771037）; 上海市浦东新区卫生系统特色专病学科建设基金（PWZzb2017-16）

Effects of Icarisid Ⅱ on proliferation and osteogenic differentiation of alveolar bone-derived stem cells in Beagle dogs

ZHANG Yi-wen¹, LUO Yuan^1,2, GUO Hua-yan³, MIAO Guo-jun³, ZHANG Ying-di³, HUANG Yuan-liang³

1.Department of Oral and Maxillofacial Surgery, School of Stomatology, Tongji University; Shanghai Engineering Research Center of Tooth Restoration and Regeneration. Shanghai 200072;
2.Department of Oral Surgery, Shanghai Stomatology Disease Centre. Shanghai 200031;
3.Department of Stomatology, Oriental Hospital, Tongji University. Shanghai 200120,China

Received:2018-12-03 Revised:2019-01-21 Online:2019-07-20 Published:2019-08-12

摘要/Abstract

摘要： 目的研究淫羊藿次苷Ⅱ（Icarisid Ⅱ,ICSII）对体外培养的比格犬颌骨间充质干细胞（alveolar bone-derived stem cells, AMSCs）增殖与成骨分化的影响。方法取比格犬颌骨骨片,体外分离培养出dAMSCs,传代至第4代,做多向分化鉴定。以10^-5、10^-6、10^-7、10^-8、10^-9 mol/L浓度的ICSII刺激dAMSCs后,取1、3、5、7 d的细胞,分别用CCK-8及碱性磷酸酶（ALP）试剂盒检测dAMSCs的增殖及ALP活性;用10^-6 mol/L的ICSII处理dAMSCs后14 d,作ALP染色,21 d时作茜素红染色,以判断ICSII对细胞分化及矿化能力的影响。在成骨诱导及10^-6 mol/L的ICSII药物诱导第4、8天后,用蛋白免疫印迹试验分析成骨相关蛋白OSX、Runx-2及OCN的表达量;在诱导第6天时,利用RT-PCR分析成骨相关基因OSX、bFGF、Runx-2及OCN的表达情况。采用SPSS 22.0软件包进行统计分析。结果原代培养的dAMSCs贴壁生长,呈梭形,具备多向分化能力;ICSII在不同浓度下均可促进dAMSCs的增殖,其ALP活性亦有提高,且在实验浓度时可观察到钙结节形成。各成骨相关基因在ICSII诱导后表达量有不同程度增加,均高于完全培养基组。随着时间延长,加药组Runx-2蛋白表达量有所下降, OCN蛋白表达量逐渐增多,与对照组差距加大;OSX的蛋白表达量虽高于对照组,但无统计学差异。结论 ICSII可促进dAMSCs的增殖及成骨分化。

关键词: 淫羊藿次苷Ⅱ, 颌骨间充质干细胞, 比格犬, 增殖, 分化

Abstract: PURPOSE: To investigate the effect of Icarisid Ⅱon proliferation and osteogenic differentiation of alveolar bone-derived stem cells(AMSCs) in Beagle dogs. Methods: Alveolar bone-derived stem cells were isolated and cultured from the jaw bone of Beagle dogs in vitro. After dAMSCs were stimulated by ICSII at 10^-5, 10^-6, 10^-7, 10^-8 and 10^-9 mol/L, the proliferation and alkaline phosphatase (ACP) activity of dAMSCs were detected by CCK-8 and ALP kit at 1, 3, 5 and 7 days. ALP staining, Alizarin red staining, Western blot and RT-PCR were performed at different time points to evaluate the osteogenic capacity of cells. SPSS22.0 software package was used for statistical analysis. Results: Primary dAMSCs adhered to the wall, showing spindle shape and multidirectional differentiation ability. ICSII can promote the proliferation of dAMSCs at different concentrations. The results showed that ALP activity was also increased, and the formation of calcium nodules was observed at the experimental concentration. The expression levels of each osteogenic gene increased, which were significantly higher than that of the complete medium group. With the extension of time, runx-2 protein expression decreased in the experimental group, OCN protein expression gradually increased, and the gap between the experimental group and the control group widened. OSX protein expression was higher than that of the control group, but there was no significant difference. Conclusions: ICSII can promote the proliferation and osteogenic differentiation of dAMSCs.

Key words: Icarisid Ⅱ, Alveolar bone-derived stem cells, Beagle dogs, Proliferation, Osteogenic differentiation

中图分类号:

R782.2

张意闻, 罗远, 郭华艳, 张迎娣, 缪国俊, 黄远亮. 淫羊藿次苷对犬颌骨间充质干细胞增殖与成骨分化的影响[J]. 中国口腔颌面外科杂志, 2019, 17(4): 304-310.

ZHANG Yi-wen, LUO Yuan, GUO Hua-yan, MIAO Guo-jun, ZHANG Ying-di, HUANG Yuan-liang. Effects of Icarisid Ⅱ on proliferation and osteogenic differentiation of alveolar bone-derived stem cells in Beagle dogs[J]. China J Oral Maxillofac Surg, 2019, 17(4): 304-310.

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淫羊藿次苷对犬颌骨间充质干细胞增殖与成骨分化的影响

Effects of Icarisid Ⅱ on proliferation and osteogenic differentiation of alveolar bone-derived stem cells in Beagle dogs

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