Experimental Study On Tissue Engineering Adipose Constructed By Adipose Cells Derived From Mesenchymal Stem Cells Transfected With Human HGF Gene And PLGA

Objective: 1. To investigate the biological characteristics of mesenchymal stem cells. 2. To discuss the transfer efficiency, HGF expression and the influence on bioactivity in MSCs-derived adipose cell via a recombinant adenovirus carrying human hepatocyte growth factor gene (Ad-HGF). 3. To study the feasibility about tissue engineer adipose grafts, which was constructed by adipose cells transfected HGF gene and PLGA ,and the effect of HGF on survival of those.Methods: 1. The MSCs of the Wistar rats were isolated with density gradient centrifugation, observed cell generation cycle by flow cytometry and induced to differentiate into adipose cells and osteoblast.2. The 3rd passage MSCs were induced to differentiate into adipose cells. They were transfected with Ad-GFP (the recombinant adenovirus carrying green fluorescence protein gene ) at the different MOI(50,100, 150, 200).At 48, 72, 96h after transfecting, the transfer efficiency was evaluated by flow cytometry and the best MOI would be found out. The adipose cells were transfected with Ad-HGF at the best MOI. HGF expression were evaluated by ELISA. The biological characteristics of adipose cells were observed by MTT.3. MSCs of male Wistar rats were collected and cultured in vitro,induced to differentiate into adipose cells. The adipose cells were transfected with Ad-GFP or Ad-HGF, and then seeded into PLGA scaffold. The Wistar female rats were randomly divided into 3 groups:group A(adipose cells-PLGA),group B(adipose cells transfected Ad-HGF-PLGA),group C(adipose cells ransfected Ad-GFP-PLGA).Then they were transplanted between the muscles of the rats' legs. Fat graft were obtained on 3,5,7,14, 28,60 days after transplantation. Absorption of fat graft and collagen proliferation were examined by electon microscope and pathology staining. The expressions of HGF and CD34 in transplanted fat tissue were detected by immunohistochemistry.Results: 1. The MSCs isolated with density gradient centrifugation displayed fibroblast-like morphology. Cell cycle studies of the MSCs that the percentage of cells on G₀/G₁ and S+G₂+M were 79.3% and 20.7% respectively. The MSCs could be induced into adipose cells and osteoblast.2. At 72h after transfecting, the transfer efficiency of Ad-GFP for the adipose cells reached to 65.39%(the highest value)at 100 MOI, and HGF expression reached the highest level(91.62ng/10⁶cells). The bioactivity of adipose cells had not to do with ransfecting.3. Compared with other two groups, at 3,7,14 days post-transplantation, the expression of HGF in group B showed statistics significance(p<0.05). Microvessel quantity in group B was much more than that in group A and group C at 3, 7, 14, 28, 60 days post-transplantation. At 28, 60 days post-transplantation, group B putresced slightly.Conclusion: 1. MSCs can be induced into adipose cells, so it can be as derived cells of adipose tissue engineer. 2.The recombinant adenovirus can transfect MSCs-derived adipose cells. The transfected cells could efficiently express the aim gene. Transfection will not influence the subsistence of the adipose cells. 3. It is feasible to constitute tissue engineer adipose grafts using adipose cells transfected with Ad-HGF and PLGA. The grafts can secrete HGF, promote angiogenesis and reduce the absorption of adipose grafts.