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Published on: June 3, 2016
[Experimental expression of HIF-1α in rabbit adipose-derived mesenchymal stem cells by dynamic compression plus IGF-1
Jun Yang1, Chuan-hui Zhang, Jian-jun Li
1Shengjing Hospital, China Medical University, Shenyang 110004, China. yangj1@sj-hospital.org
Objective:
To investigate the effects on HIF-1α expression of rabbit adipose-derived mesenchymal stem cells (ADSCs) by dynamic compression plus IGF-1 gene transfection and explore the mechanism of promoting chondrogenesis by HIF-1α.
Methods:
The ADSCs were harvested after the digestion of typeIcollagenase and transfection with pcDNA3.1-IGF-1. And then the cells were seeded onto chitosan/gelatin scaffolds with a density of 5 × 10(7) cells/ml and divided into groups: Group A (control), non-transfected ADSCs; Group B (IGF-1), hIGF-1 gene transfected ADSCs; Group C (loading), untransfected ADSCs with stimulation of compressive loading; Group D (loading + IGF-1), hIGF-1 gene transfected ADSCs and loading stimulation. The dynamic compression was carried out with a bio-reactor at a frequency of 0.1 Hz and a sinusoidal strain amplitude of 2% (2% at 0.1 Hz). The dynamic load was performed every 20 minutes, 4 hours daily. After 7 days, morphological observation was performed. The MTT assay was used to detect the cell proliferation. Meanwhile the CM-DiI cell-labeling solution was used to observe the distribution of cells. The total amount of GAG and the expression of related genes of IGF-1, HIF-1α, type II collagen (COL II) and Sox-9 were quantified.
Results:
The best morphology was found in loading+ IGF-1 group. The results of proliferating capacity were: Group A < C < B < D (P < 0.01); Dil fluorescence showed that the cells were well-distributed in Group D. Meanwhile, the content of total GAG and the expression of related genes demonstrated: Group A < C < B < D (P < 0.01). And the combined effect was more significant than either alone.
Conclusion:
In three-dimensional culture conditions, dynamic compression plus IGF-1 transfection can significantly enhance the level of autocrine IGF-1. Both of two experimental intervention factors can significantly up-regulate the mRNA level of HIF-1α. And they exert a synergistic effect. HIF-1α plays an important role in promoting the chondrogenesis and the synthesis and secretion of extra-cellular matrix.
