Experimental Study On Chondrocyte Differentiation Of Rabbit Adipose Mesenchymal Stem Cells By Gene Transfection Of Insulinlike Growth Factor-1

PurposeArticular cartilage is vulnerable to injury and has a limited capacity for self-repair. Experimental approaches toward treatment of damaged articular cartilage have increasingly focused on cell-based therapies.In this regard,Adipose Tissue-derived Stem Cells(ADSCs)provide an attractive alternative to mature chondrocytes that must be isolated from a very limited supply of healthy articular cartilage.ADSCs can be obtained relatively easily from fat tissue and have the capacity for differentiation into the cell types characteristic of various mesenchymal tissues,including cartilage and bone.Delivery of ADSCs to cartilaginous lesions has not yielded satisfactory regeneration of articular cartilage.One possible problem is that there is insufficient local stimulation of the implanted cells by the protein factors necessary to drive differentiation in vivo.Gene transfer might be adapted as a means to provide sustained synthesis of bioactive transgene products within cartilaginous lesions;the delivery of the appropriate stimulatory factors in this manner may enable synthesis of an improved cartilaginous repair tissue.The development of in vitro systems of chondrogenesis has been important to the identification of protein factors that can promote chondrocyte differentiation of ADSCs and improved cartilage repair in vivo.Related studies have been useful to the elucidation of the chondrogenic potential of other growth factors, including TGF-β1,TGF-β2,TGF-β3,Fibroblast growth factor(FGF),bone morphogenetic protein-2(BMP-2),BMP-6,and insulin-like growth factor-1(IGF-1). Here,we report that delivery of IGF-1&BMP-2 in order can induce chondrogenesis of ADSCs in aggregate culture.Materials and methods1.Study of the isolation culture and biological characteristics of rabbit Adipose Tissue-derived Stem Cells.â‘ Fat tissue aspirate of New Zealand white rabbits was digested by type I collagenase and cultured in plastic culture bottles.â‘¡osteoblast-differentiation of rabbit adipose mesenchymal stem cells in vitro.â‘¢The cells were examined by invert microscope.2.The effect of Insulinlike Growth Factor-1 gene transfer on the proliferation of rabbit Adipose Tissue-derived Stem Cells and its chondrocyte differentiation potential.â‘ Amplification and purification of plasmid DNA of pcDNA3.1-IGF-1.â‘¡ADSCs was transfected by IGF-1 gene and identificated the expression of IGF-1.â‘¢The cells were examined by invert microscope and the toluidine blue stain.â‘£Drawed the ADSCs growth curve which charged IGF-1,and analysized the cell-cycle by flow cytometry.⑤Collagenâ…¡was analysied by immunofluorescence.â‘¥Ultramicrostructure of ADSCs was observed by transmission electron microscope.Results1.Success of the isolation culture of rabbit Adipose Tissue-derived Stem Cells.â‘ After the digestion of collagenase I,the obtained cell mostly is the circular mononuclear cell.ADSCs had a similar long-spindle morphology and tightly arrayed to grow,showed no morphological changes in passage cultivation. â‘¡The induced rabbit adipose mesenchymal stem cells expressed the ALP and the node of calcium.â‘¢Growth curve showed the high proliferation ability of ADSCs.2.The effect of Insulinlike Growth Factor-1 gene transfer on the proliferation of rabbit Adipose Tissue-derived Stem Cells and its chondrocyte differentiation potential.â‘ Amplification and purification of plasmid DNA of pcDNA3.1-IGF-1 were success.â‘¡Stable transfection of IGF-1 to ADSCs was successful.â‘¢ADSCs-nodus was showed up after gene transfection.The result of toluidine blue was positive.â‘£The ADSCs showed a high reproductive activity after gene transfection.The proportion of S phase increased,and the proportion of G₁ phase decreased,p＜0.05.⑤ADSCs transfected with IGF-1 gene showed stable expression of collagenâ…¡.â‘¥Ultramicrostructure of ADSCs showed constructive metabolism active by transmission electron microscope.Conclusion1.The combination of digestion of collagenaseâ… and adherent culture was an effective method to isolate ADSCs from fat tissue aspirate.ADSCs had a higher proliferation ability.2.ADSCs transfected with IGF-1 gene showed stable expression of collagenâ…¡.