Abstract:

BACKGROUND: Adipose-derived mesenchymal stem cells (ADSCs) can be induced toward the chondrogenic lineages with chondrogenic medium contained transforming growth factor-β1(TGF-β₁). However, it remains concerned about the disadvantage with use of TGF-β₁ in vitro, which can induce chemotaxis, activate inflammatory cells, cause local defect and want an ideal matrix to deliver proteins. Therefore, with advanced gene-delivery techniques, TGF-β₁ can be long-lasting expressed by transduced stem cells, which is very useful for Chondrogenic Tissue Engineering.

OBJECTIVE: To master the method of construction and transfection of eukaryotic expression vector for rat transforming growth factor-β1 and to study the possibility of gene transfection to ADSCs with this vector.

METHODS: Recombining DNA techniques were applied to construct recombinant plasmid pcDNA3.1-TGF-β₁. And this plasmid was verified by restriction endonuclease mapping and DNA sequencing; Then the plasmid with TGF-β₁ gene or not was transfected into ADSCs by use of Lipofectamine^TM2000. After infection, transduced ADSCs were diluted and cultured with neomycin (G418). Gene transfer efficiency compared on the basis of green fluorescent protein expression was assessed.

RESULTS AND CONCLUSION: Digestion of the plasmid with double restriction endo- nuclease XboⅠand Hind Ⅲ showed about two specific electrophoretic strips (1.35 bp and 5.4 kb), and the sequence of the rat TGF-β₁ gene in recombinant was concorded with that reported in Genbank. There were 80 percent of the cells which were transduced, and the expressions of mRNA and protein of TGF-β₁ in ADSCs were discovered positively. These indicated that the eukaryotic expression vector for rat TGF-β₁ (i.e. pcDNA3.1-TGF-β₁) , which is able to transfect the ADSCs, can be constructed through genetic recombination.