Differentiation of human insulin-like growth factor-I transfected dipose mesenchymal stem cells to chondrocytes

Abstract

Objective To investigate the effectiveness of human insulin-like growth factor-I （hIGF- I ） in transfecting adipose-derived mesenchymal stem cells （AdMSCs） and the expression of the transfected genes and find the possibility of differentiation of ADSCs to chondrocytes after hIGF- I transfection. Methods AdMSCs were extracted from the fat tissue of the back of rabbits＇ neck and cultured in vitro by monolayer. Then, the recombinant eukaryotic expression plasmid pcDNA3.1 ＋ hIGF- I was transfected into AdMSCs by Lipofectamine 2000. The growth of the ADSCs was observed by inverted microscope at 4-72 hours after transfection, at the end of passage and after stable transfection respectively, and the transfection efficiency was calculated. The hIGF- I concentration in the supernatant was detected by enzyme-linked immunosorbent assay （ELISA）, the expression of collagen-Ⅱ by immunohistochemistry, the expression of hIGF- I mRNA by reverse transcription polymerase chain reaction （ RT- PCR）, the expression of hIGF- I protein by Western blot and the cell cycle by flow cytometry. Results AdMSCs were shaped long shuttle and adhered to the disk at the 4th hour. AdMSCs were increased and distributed in cluster at 24th hour. After transfection, AdMSCs were proliferated actively, with shortened doubling generation time. The concentration of hIGF- I in the supernatant increased slowly after transfec- tion and reached peak at 32.45 ng/ml at the 72nd hour. The hIGF- I concentration remained at 28.36 ng/ml after stable transfeetion. Immunohistoehemistry affirmed a large number of collagen-Ⅱ in the endochylema and positive expression of hIGF- I mRNA and hIGF- I protein. The percentage of AdMSCs at stage S increased after transfection, which was significantly higher than that of AdMSCs without transfection, with statistical difference （ P 〈 0.05）. Conclusion After stable transfection, AdMSCs can secrete high concentration of hIGF- I that can promote cell proliferation and differentiation of AdMSCs to chondrocytes. Key words: Insulin-like growth factor- I ; Mesenchymal stem cells; Transfection; Cell cycle ; Chondrocyte