Differentiation of human insulin-like growth factor-Ⅰ transfected dipose mesenchymal stem cells to chondrocytes
10.3760/cma.j.issn.1001-8050.2009.02.51
- VernacularTitle:人胰岛素样生长因子-Ⅰ转染脂肪间质干细胞向软骨细胞分化
- Author:
Chunhou AN
;
Quau YUAN
;
Jianjun LI
- Publication Type:Journal Article
- Keywords:
Insulin-like growth factor-Ⅰ;
Mesenchymal stem cells;
Transfection;
Cell cycle;
Chondrocyte
- From:
Chinese Journal of Trauma
2009;25(2):169-172
- CountryChina
- Language:Chinese
-
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 poDNA3.1 + hIGF-Ⅰwas 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 immnnosorbent assay (ELISA), the expression of collagen-Ⅱ by immunohistochemistry, the expression of hIGF-Ⅰ mRNA by reverse transcription polymerase chain reaction (RTPCR), 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 tranafection, AdMSCs were proliferated actively, with shortened doubling generation time. The concentration of hlGF-I in the supematant increased slowly after transfection and reached peak at 32.45 ng/ml at the 72nd hour. The hIGF-Ⅰ concentration remained at 28.36 ng/ml after stable transfectian. Immunohistochemistry 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.
