Genetic engineering is a main method to repair cartilage damage. Adipose mesenchymal stem cells (ADSCs) are ideal seed cells and characterized by abundant source, easy material collection, good immune compatibility, long-term differentiation phenotype cultured in vitro, and strong ability to transformation into chondrocytes.OBJECTIVE:
To study the effect of human insulin-like growth factor 1 (IGF-1) gene transfection on the proliferation of rabbit ADSCs.DESIGN, TIME AND SETTING:
The cytological, gene transfection, in vitro, study was performed at the Laboratory of Cell Biology, China Medical University from March 2006 to March 2007.MATERIALS:
Three adult New Zealand rabbits were supplied by Experimental Animal Center, China Medical University. The plasmid of pcDNA3.1-hIGF-1 containing total length human IGF-1 cDNA fragment was presented by Professor Xu from Jilin University.METHODS:
Subcutaneous fat from rabbit posterior neck was obtained. ADSCs were separated in vitro by college-I enzyme digestion method. Cells at passage 2 (0.5×106/well) were incubated in a 6-well plate. When 90%-95% confluency, ADSCs were randomized into non-transfection, pcDNA3.1, and pcDNA3.1-hIGF-1 groups. The plasmid of pcDNA3.1-hIGF-1 was transfected into ADSCs by using Lipofectamine 2000. The positive cell clones were selected by G418 and cultured for 4 weeks.MAIN OUTCOME MEASURES:
Expression of IGF-1 was quantified by RT-PCR and Western blot assay. Proliferation of ADSCs was determined by MTT assay and flow cytometer analysis.RESULTS:
IGF-1 mRNA and protein only expressed in ADSCs transfected with pcDNA3.1-hIGF-1. MTT assay showed that the proliferation speed of the group transfected by pcDNA3.1-hIGF-1 was faster than those of transfected by pcDNA3.1 and ADSCs without transfection. Flow cytometry demonstrated that compared with the groups of transfected by pcDNA3.1 and ADSCs without transfection, the cellular proportion in S phase of the group of transfected by pcDNA3.1-hIGF-1 was significantly increased (P < 0.05) and it in G1 phase was significantly decreased (P < 0.05).CONCLUSION:
The stable expression of hIGF in ADSCs transfected with pcDNA3.1-hIGF-1 is obtained. Gene transfection of hIGF-1 can stimulate the proliferation of ADSCs.