Muscle satellite cells transfected with acidic fibroblast growth factor gene in vitro

doi:10.3969/j.issn.2095-4344.2013.15.015

Abstract

Abstract:

BACKGROUND: It has been reported that acidic fibroblast growth factor (aFGF) not only can mediate cell division and differentiation, but also can prevent motor endplate degeneration and muscular atrophy.
OBJECTIVE: To calculate gene transfection efficiency and detect the target protein expression of muscle satellite cells which were transfected with aFGF gene, in purpose to further study the method to set up cell bank for preventing motor endplate degeneration and muscular atrophy.
METHODS: Muscle satellite cells were extracted from adult Wistar rat, purified by difference-speed adherence method and identified by immunohistochemical assay. The recombinant eukaryotic expression plasmid pEGFP-N1-aFGF was transfected into cells by LipofectamineTM2000 Reagent as experimental group. Muscle satellite cells transfected with pEGFP-N1 served as negative controls. Blank control group was set by adding transfection reagent. Inverted fluorescent microscope was applied to observe green fluorescent protein expression in the cells to calculate transfection efficiency at 24-72 hours after transpection and passaging. Western Blot of aFGF was performed to detect the target protein. Total RNA was extracted at 72 hours after transfection. Real-time fluorescent quantitative PCR was employed in order to find out the changes of cells after transfection on mRNA level.
RESULTS AND CONCLUSION: The immunohistochemical results showed that cultivated cells were muscle satellite cells. The expression of green fluorescent protein appeared as early as 6 hours after transfection, and the amount and intensity peaked at 72 hours. Green fluorescent protein was still seen in the subculture cells. Real-time fluorescent quantitative PCR proved stronger aFGF mRNA expression in the transfected cells with aFGF gene, while a little in the control groups. aFGF protein was highly expressed in the cells transfected with target gene detected by Western Blot. All the results indicate that the aFGF gene can be transfected efficiently and safely into muscle satellite cells and expressed normally, which can serve as the new seed cells for tissue engineering to prevent motor endplate degeneration and muscular atrophy.

Key words: tissue construction, tissue construction and bioactive factors, acidic fibroblast growth factor, gene, muscle satellite cells, transfection, plasmid, green fluorescent protein, motor endplate, muscular atrophy, nerve injury, denervation, provincial grants-supported paper

CLC Number:

R318

Yang Shao-an, Cai Jin-kui, Zhou Chu-song, Xiao Xiao-tao, Xiao Sha, Zou Xiao-ying. Muscle satellite cells transfected with acidic fibroblast growth factor gene in vitro[J]. Chinese Journal of Tissue Engineering Research, doi: 10.3969/j.issn.2095-4344.2013.15.015.

Figures/Tables 5

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