CRISPR系统促进脂肪干细胞成骨分化与抑制成脂分化的研究

doi:10.16431/j.cnki.1671-7236.2019.10.007

Abstract

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

CLC Number:

Q254

YANG Furu, WANG Xuezhi, YU Sijiu. Study on Stimulation of Osteogenic Differentiation and Inhibition of Adipogenic Differentiation in Adipose-derived Stem Cells via CRISPR System[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(10): 2867-2875.

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

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