中国组织工程研究 ›› 2018, Vol. 22 ›› Issue (13): 1987-1992.doi: 10.3969/j.issn.2095-4344.0493

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

补肾活血汤水提物调控Cbfal/RUNX2基因沉默骨髓间充质干细胞SP7/Osterix及碱性磷酸酶的表达

程英雄,罗毅文,王 斌,吴志方,沈 玮,罗 辉,孙世栋,黄文强   

  1. 广州中医药大学附属骨伤科医院,广东省广州市 510240
  • 修回日期:2018-03-12 出版日期:2018-05-08 发布日期:2018-05-08
  • 通讯作者: 程英雄,副主任中医师,广州中医药大学附属骨伤科医院,广东省广州市 510240
  • 作者简介:程英雄,男,1976年生,湖南省祁东县人,汉族,2010年广州中医药大学毕业,博士,副主任中医师,主要从事中医骨伤科工作。
  • 基金资助:

    广东省科技厅项目(2014A020221022)

Expression of SP7/Osterix and alkaline phosphatase in bone marrow mesenchymal stem cells with Cbfal/RUNX2 gene silencing regulated by the water extracts from Bushen Huoxue Decoction

Cheng Ying-xiong, Luo Yi-wen, Wang Bin, Wu Zhi-fang, Shen Wei, Luo Hui, Sun Shi-dong, Huang Wen-qiang   

  1. Orthopedic Hospital, Guangzhou University of Chinese Medicine, Guangzhou 510240, Guangdong Province, China
  • Revised:2018-03-12 Online:2018-05-08 Published:2018-05-08
  • Contact: Cheng Ying-xiong, Orthopedic Hospital, Guangzhou University of Chinese Medicine, Guangzhou 510240, Guangdong Province, China
  • About author:Cheng Ying-xiong, M.D., Associate chief physician, Orthopedic Hospital, Guangzhou University of Chinese Medicine, Guangzhou 510240, Guangdong Province, China
  • Supported by:

    the Project of Guangdong Provincial Science and Technology Department, No. 2014A020221022

摘要:

文章快速阅读:

文题释义:
RUNX2因子:
也称Cbfal,是调控骨形成的一种重要的转录激活因子,属于Runt结构区域家族,该基因在1997年被不同科学家在不同实验中证实为控制骨形成的关键因子,在间充质干细胞向前成骨细胞分化阶段中,RUNX2发挥了极其重要的作用。
Osterix因子:是一种由428个氨基酸组成的具有锌指基序结构域的转录因子,最早由Nakashima发现其为成骨细胞分化和骨形成所必需的转录因子之一,是成骨细胞特异性表达因子。OSX的人类同源物又称SP7,属于SP/XFL家族之一,在一项骨形态发生蛋白2诱导的成肌细胞C2C12研究中发现,该因子参与调控许多重要的成骨基因的表达活动,如骨钙素、骨涎蛋白、Ⅰ型胶原蛋白等。

 

摘要
背景:
补肾活血汤可以促进骨髓间充质干细胞成骨分化,探究分子机制利于推向临床。
目的:探讨补肾活血汤水提物对Cbfal/RUNX2基因沉默的骨髓间充质干细胞Osterix、碱性磷酸酶表达的影响。
方法:骨髓贴壁筛选法分离和培养大鼠骨髓间充质干细胞,Cbfal/RUNX2基因沉默慢病毒转染骨髓间充质干细胞。取第3代骨髓间充质干细胞(空白对照组)、慢病毒沉默骨髓间充质干细胞(沉默组)和阴性病毒载体预处理骨髓间充质干细胞(阴性对照组),以及上述3组骨髓间充质干细胞加入100 mg/L补肾活血汤水提物干预3 d后,检测RUNX2、Osterix蛋白和mRNA表达以及碱性磷酸酶活性。
结果与结论:①Cbfal/RUNX2基因沉默慢病毒转染效率达约90%;②与空白对照组和阴性对照组比较,沉默组骨髓间充质干细胞RUNX2和Osterix的蛋白及mRNA表达均下降,碱性磷酸酶活性也明显下降,差异有显著性意义(P < 0.01);③补肾活血汤水提物干预后,沉默组骨髓间充质干细胞RUNX2、Osterix的表达和碱性磷酸酶活性明显增加,差异有显著性意义(P < 0.01);④结果表明,补肾活血汤水提物可以促进骨髓间充质干细胞的RUNX2、Osterix表达,亦增加了碱性磷酸酶活性,从而促进骨髓间充质干细胞成骨分化。

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
ORCID: 0000-0001-6661-1179(程英雄)

关键词: 补肾活血汤, 成骨, 骨髓间充质干细胞, RUNX2, Osterix, 基因沉默, 碱性磷酸酶, 干细胞

Abstract:

BACKGROUND: Bushen Huoxue Decoction (BSHXD) can promote osteogenesis of bone marrow mesenchymal stem cells (BMSCs) in vitro. Exploring the molecular mechanisms involved is of clinical benefits.
OBJECTIVE: To discuss the changes in the expression of SP7/Osterix and alkaline phosphatase (ALP) in BMSCs with Cbfal/RUNX2 gene silencing regulated by the water extracts from BSHXD.
METHODS: BMSCs were isolated and cultured by the bone marrow adherent method, and BMSCs at passage 3 were used in the assay. BMSCs were transfected with nothing (blank control group), Cbfal/RUNX2 gene silencing lentivirus (silencing group), and negative viral vector (negative control group), respectively. Then, the cells were cultured in 100 mg/L BSHXD water extract, and 3 days later, the protein and mRNA expression of RUNX2 and Osterix was detected by western blot and qPCR, respectively. Activity of ALP in the BMSCs was also detected in each group.
RESULTS AND CONCLUSION: The transfection efficiency of Cbfal/RUNX2 gene silencing lentivirus was about 90%. The protein and mRNA expressions of RUNX2 and Osterix were significantly decreased in the BMSCs transfected with Cbfal/RUNX2 gene silencing lentivirus as compared with the other two groups, and so was the ALP activity (P < 0.01). After treated with the water extracts from BSHXD, the expression of RUNX2 and Osterix as well as the ALP activity in the BMSCs transfected with Cbfal/RUNX2 gene silencing lentivirus increased significantly (P < 0.01). To conclude, the water extract from the BXHXD can up-regulate the expression of RUNX2 and Osterix and the activity of ALP, thus promoting BMSCs osteogenic differentiation.  

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

Key words: Bone Marrow, Mesenchymal Stem Cells, Gene Silencing, Core Binding Factor Alpha 1 Subunit, Drugs, Chinese Herbal, Tissue Engineering

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