中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (11): 1913-1917.doi: 10.3969/j.issn.1673-8225.2011.11.004

• 骨组织构建 bone tissue construction • 上一篇    下一篇

Brg1调控重组人骨形态发生蛋白2诱导成骨细胞分化

高忠平，秦  丽，张  越，耿倩倩，孙奋勇   

  1. 暨南大学生物工程研究所，广东省广州市 510632
  • 收稿日期:2010-12-08 修回日期:2011-01-14 出版日期:2011-03-12 发布日期:2011-03-12
  • 通讯作者: 孙奋勇，博士，博士生导师，暨南大学生物工程研究所，广东省广州市510632 sunfenyong@263.net
  • 作者简介:高忠平★，男，1984年生，江西省新余市人，汉族，硕士，主要从事遗传学方面的研究。 gaozhongping2010@163.com
  • 基金资助:

    国家自然科学基金项目(81071524)资助。

Brg1 regulates bone morphogenetic protein-2 induced osteogenic differentiation

Gao Zhong-ping, Qin Li, Zhang Yue, Geng Qian-qian, Sun Fen-yong   

  1. Institute of Bioengineering, Jinan University, Guangzhou  510632, Guangdong Province, China
  • Received:2010-12-08 Revised:2011-01-14 Online:2011-03-12 Published:2011-03-12
  • Contact: Sun Fen-yong, Doctor, Doctoral supervisor, Institute of Bioengineering, Jinan University, Guangzhou 510632, Guangdong Province, China sunfenyong@263.net
  • About author:Gao Zhong-ping★, Master, Institute of Bioengineering, Jinan University, Guangzhou 510632, Guangdong Province, China gaozhongping2010@163.com
  • Supported by:

    the National Natural Science Foundation of China, No. 81071524*

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被引次数

1

摘要：

背景：Brg1是依赖ATP的染色质改变复合物的核心催化亚基，该亚基在基因的转录调控、复制、重组，骨骼肌的分化、抑制肿瘤的发生等活动中起着重要的作用。
目的：探索Brg1基因在骨形态发生蛋白2诱导成骨细胞分化过程中的调控机制。
方法：采用胶原酶消化法进行小鼠颅骨成骨细胞的原代培养；分别用0，50，200 μg/L的重组人骨形态发生蛋白2诱导原代培养的成骨细胞的分化，摸索骨形态发生蛋白2的最佳作用剂量；实时荧光定量PCR和Western blot进行骨形态发生蛋白2对Brg1的作用时间的动力学分析；实时荧光定量PCR和钙钴染色法检测敲除Brg1对骨形态发生蛋白2诱导的成骨分化的影响；构建Dlx5腺病毒重组表达载体，实时荧光定量PCR和钙钴染色法检测Brg1在骨形态发生蛋白2诱导的成骨分化过程中对Dlx5的调控作用。
结果与结论：用自行合成的重组人骨形态发生蛋白2可诱导原代培养小鼠成骨细胞分化，200 μg/L剂量有着较好的诱导分化效果；重组人骨形态发生蛋白2可诱导Brg1基因转录水平和翻译水平表达水平上调；敲除Brg1可抑制重组人骨形态发生蛋白2诱导的成骨分化；Brg1能够调控Dlx5的表达水平。说明Brg1通过调控Dlx5的表达水平调控重组人骨形态发生蛋白2诱导的小鼠成骨细胞的分化。

关键词: 成骨, 染色质重塑, Brg1, Dlx5, 骨形态发生蛋白2

Abstract:

BACKGROUND: Brg1 is an ATPase subunit of the SWI/SNF complex and plays an important role in the regulation of gene transcription, replication and recombination, skeletal muscle differentiation and inhibition carcinogenesis.
OBJECTIVE: To explore the Brg1 regulation mechanism in bone morphogenetic protein 2 (BMP-2)-induced osteogenic differentiation.
METHODS: Mouse calvarial osteoblasts were primarily cultured through collagenase digestion and induced differentiation using 0, 50 or 200 μg/L BMP2 to select the optimal dose. Transcription time dynamics analysis of Brg1 was enforced by RT-PCR and Western blot after BMP-2 treatments; ALP staining was used to evaluate the capability of osteogenic differentiation after BMP-2 treatments. The role of BMP-2 on Brg1 mRNA expression level was detected by quantitative RT-PCR and the protein level was detected by Western Blot. Effect of Brg1 on BMP-2-induced differentiation was analyzed by Brg1-siRNA; Ad-Dlx5 was obtained by recombinant adenovirus.
RESULTS AND CONCLUSION: BMP-2 could induce the osteogenic differentiation of primarily cultured mouse osteoblast, and the optimal BMP2 induction condition was 200 μg/L. The expression of Brg1 was up-regulated by BMP-2 in primarily cultured mouse osteoblasts. BMP-2 induced the osteogenic differentiation was inhibited by Brg1-siRNA. The expression of Dlx5 was regulated by Brg1. Brg1 could regulate the BMP-2-induced osteogenic differentiation of primarily cultured mouse osteoblast through regulating the expression of Dlx5.

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