中国畜牧兽医 ›› 2019, Vol. 46 ›› Issue (10): 2867-2875.doi: 10.16431/j.cnki.1671-7236.2019.10.007

• 生理生化 • 上一篇    下一篇

CRISPR系统促进脂肪干细胞成骨分化与抑制成脂分化的研究

杨馥如1,2, 王学智3, 余四九1   

  1. 1. 甘肃农业大学动物医学院, 兰州 730050;
    2. 中国农业科学院上海兽医研究所, 上海 200241;
    3. 中国农业科学院兰州畜牧与兽药研究所, 兰州 730050
  • 修回日期:2019-06-28 出版日期:2019-10-20 发布日期:2019-10-21
  • 通讯作者: 余四九 E-mail:yusj@gsau.edu.cn
  • 作者简介:杨馥如(1986-),女,甘肃兰州人,硕士,研究方向:动物胚胎工程,E-mail:yangfuru@shvri.ac.cn
  • 基金资助:
    国家科技基础工作专项(2013FY110600-6号)

Study on Stimulation of Osteogenic Differentiation and Inhibition of Adipogenic Differentiation in Adipose-derived Stem Cells via CRISPR System

YANG Furu1,2, WANG Xuezhi3, YU Sijiu1   

  1. 1. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730050, China;
    2. Shanghai Veterinary Research Institute of CAAS, Shanghai 200241, China;
    3. Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS, Lanzhou 730050, China
  • Revised:2019-06-28 Online:2019-10-20 Published:2019-10-21

摘要: 本研究旨在优化组织培养法分离小鼠脂肪间充质干细胞(adipose-derived stem cells,ASCs),为研究成骨分化和成脂分化在间充质干细胞分化过程中的相互影响奠定基础。通过细胞形态学观察、细胞生长曲线和流式仪器检测所分离获得的间充质干细胞的特性,利用CRISPR-dCas9系统在快速促进间充质干细胞成骨分化的前提下观察其对成脂分化的影响,并通过生化染色、实时荧光定量PCR和免疫细胞学等手段进行分析。结果显示,接种3~5 d后可见细胞从组织块周围爬出,光镜下可见细胞形态多为成纤维细胞样的梭形细胞,且形态单一均匀,具有较高的爬出率,可以大大提高脂肪间充质干细胞的分离效率;通过CRISPR-dCas9系统激活Runx2和Osterix基因后可以促进间充质干细胞的成骨分化,实时荧光定量PCR及油红O染色结果显示,CRISPR-dCas9系统可以同时抑制间充质干细胞的成脂分化;通过CRISPR-dCas9-KRAB系统同时抑制成骨相关基因Runx2和Osterix后可以促进成脂分化。本研究利用组织贴壁法成功获得了高纯度的脂肪间充质干细胞,具有间充质干细胞的特性和分化能力;利用CRISPR系统可以同时过表达Runx2和Osterix两个基因,可以在进成骨分化的同时抑制成脂分化,表明成脂分化和成骨分化的相关性,为基因编辑在间充质干细胞诱导分化和临床应用方面提供了新的思路和方法。

关键词: 脂肪间充质干细胞(ASCs); CRISPR系统; 成骨诱导分化; 成脂诱导分化

Abstract: The aim of this study was to modify the method of isolating mouse adipose-derived stem cells (ASCs),which laid a foundation for studying the interaction between osteogenic differentiation and adipogenic differentiation in the process of mesenchymal stem cells differentiation.The characteristics of mesenchymal stem cells were verified through studying the cell morphology,cell growth curve and flow cytometry.The effect of CRISPR-dCas9 system on the adipogenic differentiation of mesenchymal stem cells was observed on the premise of rapid promotion of osteogenic differentiation of mesenchymal stem cells.The results were analyzed by biochemical staining,Real-time quantitative PCR and immunocytology methods,respectively.The results showed that mesenchymal stem cells could be isolated from the adipose-tissue after 3-5 d,the cell morphology was mostly fibroblast-like spindle,and the morphology had high purity and homogeneous,with a high adherent rate,this method could greatly improve the isolation efficiency of mesenchymal stem cells.The results indicated that activation of Runx2 and Osterix genes by CRISPR-dCas9 system promoted osteogenic differentiation and meanwhile inhibited adipogenic differentiation,and further inhibition of Runx2 and Osterix genes related to osteogenesis by CRISPR-dCas9-KRAB system promoted adipogenic differentiation.In this study,high purity adipose mesenchymal stem cells were successfully obtained by tissue adherence method,which possessed the characteristics and differentiation ability of mesenchymal stem cells.It was confirmed that Runx2 and Osterix genes could overexpress simultaneously using CRISPR system,and inhibit adipogenic differentiation as well as osteogenic differentiation,which indicated the correlation between adipogenic differentiation and osteogenic differentiation,and provided a new idea and method for gene editing in mesenchymal stem cells induction and clinical application.

Key words: adipose-derived stem cells (ASCs); CRISPR system; osteogenic differentiation; adipogenic differentiation

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