Chondrogenic Differentiation Of Rabbit Mesenchymal Stem Cells Following Gene Transfer Of IGF-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, adult mesenchymal stem cells ( MSCs) provide an attractive alternative to mature chondrocytes that must be isolated from a very limited supply of healthy articular cartilage. MSCs can be obtained relatively easily from bone marrow and other tissue sources and have the capacity for differentiation into the cell types characteristic of various mesenchymal tissues, including cartilage and bone. Delivery of MSCs 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 chon-drogenesis has been important to the identification of protein factors that can promote chondrocyte differentiation of adult MSCs 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, Fi-broblast growth factor(FGF) ,bone morphogenetic protein -2(BMP-2) , BMP- 6, and insulinlike growth factor - 1 (IGF -1). Here, we report that deliveryof IGF - 1 can induce chondrogenesis of MSCs in aggregate culture.Methods1. Study of the isolation culture and biological characteristics of rabbit bone marrow - derived mesenchymal stem cells.a. Bone marrow aspirate of Japan white rabbits was density gradient centri-fuged and cultured in plastic culture bottles.b. Flow cytometry analysis of cell surface markers in MSCs.c. The cells were examined by invert microscope and the growth curve was drawn.2. Construction of expression vectors of human insulinlike growth factor - 1 gene and green fluorescent protein with two multiclone sites.a. Amplification and purification of plasmid DNA of pcDNA3.1 - IGF - 1.b. Identification of plasmid DNA of pcDNA3.1 - IGF - 1.c. Construction of recombinant pcGI.d. Identification of recombinant pcGI.3. The effect of insulinlike growth factor - 1 gene transfer on the proliferation of rabbit bone marrow mesenchymal stem cells and its chondrocyte differentiation potential.a. MSCs was transfected by IGF - 1 gene and its identification.b. MSCs was observed after being transfected and transfection efficiency was calculated.c. Western blot analysis were used to measure collagen II and IGF -1 expressing in MSCs before and after being transfected by IGF -1 gene.Results1. Success of the isolation culture of rabbit bone marrow - derived mesenchymal stem cells.a. After density gradient centrifugation, the obtained cell mostly is the circular mononuclear cell. MSCs had a similar long - spindle morphology and tightly arrayed to grow, showed no morphological changes in passage cultivation.b. Flow cytometry analysis of cell surface markers in MSCs showed that markers of CD105, CD166, CD29 and CD44 were positive and markers of CD34, CD14 and CD45 were negative.c. Growth curve showed proliferation ability of MSCs was similar at passage 1, passage 3 and passage 5.2. IGF - 1 eukaryotic expression vector has been successfully constructed with GFP.a. Amplification and purification of plasmid DNA of pcDNA3. 1 - IGF - 1 were success.b. Enzyme digestion analysis of pcDNA3.1 - IGF - 1 proved that plasmid of pcDNA3. 1 - IGF - 1 contained IGF - 1 cDNA fragment.c. Sequence data of IGF -1 cDNA fragment were in accordance with those reported in literature.d. Enzyme digestion and agarose gel electrophoresis analysis revealed that pcGI vector contained correct GFP and IGF -1 cDNA.3. MSCs transfected with IGF - 1 gene showed marked increase of IGF - 1 expression and stable expression of collagen II.a. Eukaryotic expression vector pcDNA3. 1 - IGF - 1 plasmid was tranfect-ed into MSCs with the help of lipofectamine. Green fluorescence was detected in successfully transfected MSCs with fluorescence microscopy.b. Transfection efficiency was(30 ±2.5) % after 24 hours.c. Western blot analysis showed that gray value of transfected group is higher than non - transfected group or transfected group by vector.Conclusion1. The combination of density gradient centrifugation and adherent culture is an effective method to isolate MSCs from bone marrow aspirate. MSCs has higherproliferation ability and can be used in tissue engineering.2. IGF - 1 eukaryotic expression vector has been successfully constructed with GFP.3. MSCs transfected with IGF -1 gene showed marked increase of IGF -1 expression and stable expression of collagen II...