Effects of human insulin-like growth factor 1 gene trasfection on adipose- derived stem cells

doi:10.3969/j.issn.2095-4344.2013.06.012

Abstract

Abstract:

BACKGROUND: Human insulin-like growth factor 1 gene produces effects on the proliferation and differentiation of adipose-derived stem cells.
OBJECTIVE: To investigate the possible effects of human insulin-like growth factor 1 gene transfection on the adipose-derived stem cells cultured in vitro.
METHODS: The cDNA for human insulin-like growth factor 1 was cloned into the recombinant eukaryotic expression plasmid pIRES2-EGFP-hIGF-1. The adipose-derived stem cells were derived from adipose tissue and cultured in vitro, and then the plasmid was transfected into cells by Lipofectamine 2000. After gene transfection, changes in cell proliferation and morphology were observed. Through the use of inverted fluorescent microscopy, the marker gene coding enhanced green fluorescent protein was observed and the transfection efficiency was determined. The human insulin growth factor 1 concentration in the supernatant fluids was measured by ELISA. The expression of human insulin-like growth factor 1 was detected by immunohistochemical staining and RT-PCR. The changes in cell cycle before and after transfection were assessed by flow cytometry.
RESULTS AND CONCLUSION: The recombinant eukaryotic expression plasmid pIRES2-EGFP-hIGF-1 was confirmed by gene sequencing and enzyme digestion. The adipose-derived stem cells cultured in vitro were in multiple shapes. The expression of enhanced green fluorescent protein was detected for the first time at 6 hours after transfection and peaked at 60 hours, with transfection efficacy of 16±3%. The concentration of insulin-like growth factor 1 in the supernatant fluid was 22.65 μg/L at 60 hours after transfection. Human insulin growth factor 1 expression was detected by immunohistochemical staining and RT-PCR. Division and proliferation of the cells were accelerated after gene transfection, leading to the decrease of the population doubling time, and the increase of the percentage of stem cells in the S stage. These results indicate that human insulin growth factor 1 gene can be transfected efficiently into the adipose-derived stem cells by liposome and human insulin-like growth factor 1 protein can be secreted into the supernatant fluid, therefore accelerating the proliferation of the cells.

Key words: stem cells, adipose-derived stem cells, insulin-like growth factor, gene, transfection, cell proliferation, National Natural Science Foundation of China, stem cell photographs-containing paper

CLC Number:

R394.2

Zhang Hai-ning, Ding Chang-rong, Lü Cheng-yu, Wang Ying-zhen, Zhou Feng, Xu Zong-yao. Effects of human insulin-like growth factor 1 gene trasfection on adipose- derived stem cells[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(6): 1018-1023.

Figures/Tables 4

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