04 March 2015 : Laboratory Research
Med Sci Monit 2015; 21:681-688
BACKGROUND: Treatment strategies for meniscal injury are shifting from meniscectomy to repair, especially cell-based therapy. Delivering selected genes to donor cells can modify differentiation and proliferation. Efficiency of gene transfection and expression may relate to cell type.
MATERIAL AND METHODS: Full-length hIGF-1 cDNA was cloned into eukaryotic expression vector by PCR. Human BMSCs and meniscal fibrochondrocytes were isolated and cultured in vitro and hIGF-1 gene was transfected by FuGene 6. Expression of EGFP and hIGF-1 were determined. Biological activity of the hIGF-1 in medium was assessed by MTT chromatometry. Real-time quantitative PCR and Western blot were used to assess the expression of exogenous genes. Efficacy of gene transfection was detected by immunohistochemistry staining and flow cytometry.
RESULTS: Sequences of hIGF-1 were verified by sequence analysis. Expression of EGFP increased gradually and reached peak intensity 48 h after transfection. Transfection efficiency of BMSCs was higher than meniscal fibrochondrocytes. The population doubling time was decreased in both cell types. Peak concentration of hIGF-1 in the medium of BMSCs and meniscal cells was 32.5±4.8 ng/ml and 24.5±4.6 ng/ml, respectively. Secreted hIGF-1 possessed the ability to enhance proliferation of the cell line. Results of qPCR and Western blot confirmed the expression of hIGF-1. Type II collagen appeared within the cells, and percentage of cells in S stage was increased in both cell types after transfection.
CONCLUSIONS: hIGF-1 cDNA can be transfected into BMSCs and meniscal fibrochondrocytes, resulting in gene expression. Expression efficiency in BMSCs was higher than that in fibrochondrocytes.
Keywords: Chondrocytes - metabolism, Cell Shape, Blotting, Western, Genetic Vectors, Green Fluorescent Proteins - metabolism, Insulin-Like Growth Factor I - genetics, Menisci, Tibial - cytology, Mesenchymal Stromal Cells - metabolism, Real-Time Polymerase Chain Reaction, Recombination, Genetic - genetics
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