吉林大学学报(医学版) ›› 2017, Vol. 43 ›› Issue (06): 1130-1136.doi: 10.13481/j.1671-587x.20170612

• 研究基础 • 上一篇    下一篇

银杏叶提取物对破骨细胞分化和骨吸收的作用及其机制

王俊妹1, 孙千月2, 吴哲1,2, 李彤1, 李婧1   

  1. 1. 吉林大学口腔医院修复科, 吉林 长春 130021;
    2. 广州医科大学附属口腔医院修复科, 广东 广州 510500
  • 收稿日期:2017-05-18 出版日期:2017-11-28 发布日期:2017-12-01
  • 通讯作者: 吴哲,教授,硕士研究生导师(Tel:020-61350515,E-mail:zhewudentist@gzhmu.edu.cn) E-mail:zhewudentist@gzhmu.edu.cn
  • 作者简介:王俊妹(1992-),女,河南省驻马店市人,在读医学硕士,主要从事口腔修复学方面的研究。
  • 基金资助:
    广东省教育厅科研项目资助课题(B16036078);吉林省科技厅科研项目资助课题(20140204022SF);广州医科大学青年科研项目资助课题(2015A32)

Effects of Ginkgo Bitoba extract on differentiation and bone resorption ofosteoclasts and their mechanisms

WANG Junmei1, SUN Qinyue2, WU Zhe1,2, LI Tong1, LI Jing1   

  1. 1. Department of Prosthodontics, Stomatology Hospital, Jilin University, Changchun 130021, China;
    2. Department of Prosthodontics, Affiliated Stomatology Hospital, Guangzhou Medical University, Guangzhou 510500, China
  • Received:2017-05-18 Online:2017-11-28 Published:2017-12-01

摘要: 目的:探讨不同浓度银杏叶提取物(GBE)对破骨胞分化和骨吸收的作用,并阐明其作用机制。方法:体外培养RAW264.7细胞,采用核因子κB受体活化因子配体(RANKL)和不同浓度GBE处理细胞,分为空白对照组(0μg·L-1 RANKL)、RANKL组(100 μg·L-1RANKL)和RANKL+75mg·L-1 GBE和RANKL+150 mg·L-1 GBE组。抗酒石酸酸性染色(TRAP)法观察各组破骨细胞的形态及数量,骨吸收陷窝面积评估GBE对破骨细胞骨吸收能力的影响,流式细胞术检测细胞凋亡率及细胞周期,RT-PCR法检测RAW264.7细胞中破骨细胞相关基因活化T细胞核因子c1(NFATc1)、树突状细胞特异性跨膜蛋白(DC-STAMP)、组织蛋白酶K (Cathepsin K)、基质金属蛋白酶9(MMP-9)、B淋巴细胞瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、P27和细胞周期蛋白D1(Cyclin-D1)的表达水平。结果:与空白对照组比较,RANKL组破骨细胞的数量明显增加(P < 0.05);与RANKL组比较,RANKL+75 mg·L-1 GBE和RANKL+150 mg·L-1 GBE组破骨细胞数量明显降低(P < 0.05)。与空白对照组比较,RANKL组骨片中骨吸收陷窝面积明显升高(P < 0.05);与RANKL组比较,RANKL+75 mg·L-1 GBE和RANKL+150 mg·L-1 GBE组骨片中骨吸收陷窝面积明显降低(P < 0.05)。与空白对照组比较,75和150 mg·L-1 GBE组RAW264.7细胞凋亡率升高(P < 0.05),RAW264.7细胞中Bcl-2基因表达水平明显降低(P < 0.05),Bax基因表达水平明显升高(P < 0.05)。与RANKL组比较,RANKL+75 mg·L-1 GBE和RANKL+150 mg·L-1 GBE组RAW264.7细胞G0-G1期阻滞明显缩短(P < 0.05);RAW264.7细胞中P27基因表达水平明显降低(P < 0.05),Cyclin-D1基因表达水平明显升高(P < 0.05)。与空白对照组比较,RANKL组RAW264.7细胞中破骨细胞相关基因NFATc1、DC-STAMP、Cathepsin K和MMP-9表达水平明显升高(P < 0.05);与RANKL组比较,RANKL+75 mg·L-1 GBE和RANKL+150 mg·L-1 GBE组RAW264.7细胞中破骨细胞相关基因NFATc1、DC-STAMP、Cathepsin K和MMP-9表达水平明显降低(P < 0.05)。结论:GBE可抑制破骨细胞分化和骨吸收能力,其机制可能与促进RAW264.7细胞凋亡、缩短RANKL诱导的RAW264.7细胞的G0-G1期有关。

关键词: 银杏叶提取物, 破骨细胞, 细胞周期, 细胞凋亡

Abstract: Objective:To study the effects of Ginkgo Biloba extract(GBE) on the differentiation and bone resorptionof the osteoclasts, and to clarify their mechanisms. Methods:The RAW264.7 cells were cultured in vitro, then were treated with receptor activator for nuclear factor-κB ligand (RANKL) and different concentrations of GBE. The cells were divided into blank control group(0 μg·L-1 RANKL), RANKL group(100 μg·L-1 RANKL), RANKL+75 mg·L-1 GBE group,and RANKL+150 mg·L-1 GBE group.The morphology and number of osteoclasts were assessed with TRAP staining assay, and bone resorption of GBE was examined with bone resorption pits assay. Flow cytometry was applied to analyze the the apoptotic rate of RAW264.7 cells and the cell cycle;the expression levels of nuclear factor of activated T cells 1 (NFATc1), DC-STAMP, Casthepin K,matrix metalloprotein 9(MMP-9), Bcl-2, Bax,P27 and Cyclin-D1 in the RAW264.7 cells were analyzed by RT-PCR method. Results:Compared with blank control group, the number of osteoclasts in RANKL group were significantly increased(P<0.01); compared with RANKL group,the number of osteoclasts in RANKL+75 mg·L-1 GBE and RANKL+150 mg·L-1 GBE groups were significantly decreased (P<0.05). Compared with blank control group,the area of bone resorption pit of bone slice in RANKL group was significantly increased(P<0.05);compared with RANKL group, the areas of bone resorption pit of bone slice in RANKL+75 mg·L-1 GBE and RANK+150 mg·L-1 GBE groups were significantly decreased (P<0.05). Compared with blank control group, the apoptotic rates of the RAW264.7 cells in 75 and 150 mg·L-1 GBE groups were increased, and the expression levels of Bcl-2 in the RAW264.7 cells were significantly decreased and the expression levels of Bax were significantly increased (P<0.05). Compared with RANKL group, the G0-G1 phase arrest of the RAW264.7 cells in RANKL+75 mg·L-1 GBE and RANKL 150 mg·L-1 GBE groups were shortened;the expression levels of P27 in the RAW264.7 cells were significantly decreased and the expression levels of Cyclin-D1 were significantly increased (P<0.05). Compared with blank control group, the expression levels of NFATc1, DC-STAMP, Casthepin K and MMP-9 in the RAW264.7 cells in RANKL group were significantly increased(P<0.05); compared with RANKL group, the expression levels of NFATc1, DC-STAMP, Casthepin K and MMP-9 in the RAW264.7 cells in RANKL+75 mg·L-1 GBE and RANKL+150 mg·L-1 GBE groups were significantly decreased (P<0.05). Conclusion:GBE could inhibit the differentiation and bone resorption of the osteoclasts, and their mechanisms may be related to promoting the apoptosis of RAW264.7 and shortening the G0-G1 phase of the RAW264.7 cells.

Key words: Ginkgo Biloba extract, osteoclast, cell cycle, apoptosis

中图分类号: 

  • R329.28