The Effect Of Adenovirus-mediated Hepatocyte Growth Factor On Promoting The Survival Of Fat Transplantation

The autotransplantated adipose tissue is an ideal material to restore congenital or traumatic defects because it is abundant,autologous and.without allergic reactions. Nevertheless adipocytes are vulnerable for ischemia,some adipocytes would be necrosis if the recipient site can not be revascularization in short time,which result in reducing the fat tissue survival.Therefore,the prompt establishment of revascularization plays a key factor for the survival of implanted adipose.Hepatocyte growth factor(HGF) known as the most powerful vascular growth factor,appears to induce angiogensis and inhibit the formation of scar,stimulates the proliferation of multiple cells.In this research,we use recombinant adenovirus vector encoding of HGF in adipose tissue transplantation to investigate the effect of HGF on the survival of fat transplantation and the quantification of revascularization of transplanted adipose tissue.Objective:â‘ To observe the efficiency of infection Ad-HGF on adipose derived stem cells and to prove whether the valid HGF can appear after infection and the multiplicity of infection.â‘¡To observe the effect of Ad-HGF in revascularization and the survival of implanted adipose in nude mice.Research methods and procedureswe use the digestion separation method and the attaching-wall characteristic of the adipose-derived stem cells to separate the human adipose-derived stem cells.The adherent cells displayed themselves as fibroblast in morphology.The primary cultured cells fusion can arrive to 70%-80%in 7-10days.We use the vector of adenovirus(Ad-GFP) which carries the GFP gene to infect the adipose-derived stem cells,and the GFP acts as the indicating gene to determine the infection efficiency of recombinant adenovirus to adipose-derived stem cells. Immunofluorescence studies show that the infection efficiency reaches 95.64%with the infection intensity MOI=300pfu/cell.we use the Ad-HGF of the same MOI to infect adipose-derived stem cells and use HGF-ELISA to detect HGF as expression-secretion.Result shows that the infected HGF can be highly expressed in 48H,which indicate adenoviral can meditate the expression of HGF in adipose-derived stem cells effectively.Based on above experiments,we make the experimental research to determine the effect of Ad-HGF on fat free transplantation in nude mice.We use 30 6-week old nude mice in three random groups with 10 in each group.Each mouse is injected 1 ml of the fat tissue extracted from human abdomen in the scalp.Experimental group: add Ad-HGF in fat graft.Control group 1:add Ad-GPF in fat graft,Control Group 2: add normal saline in fat graft.The nude mice were bred 10 weeks after the fat injection.In 1day,4 days,8 days and 15 days after the injection,we take blood sampling from the tail of nude mice and use ELISA to detect the HGF serum concentration.The result shows that the serum level is much higher in experimental group than that in control groups and it indicates the serum level augments effectively after the infection of HGF gene in adipose-derived stem cells.After 10 weeks of breeding,we weigh,kill the mice and anatomize the adipose graft.The comparison of graft weight is made as well as the HE staining of the pathological section and Immunohistochemical staining ofâ…§factor in these grafts. The grafts from the experimental group are much heavier than other two control groups and between two control groups there is no obvious difference.It proves that HGF can raise the survival rate and reduce the absorption of the implanted adipose significantly.Pathological analysis shows that:much less vacuole is formed in the experimental group which can indicate that HGF promotes the growth of adipose tissues.The immunohistochemistry shows that vascular density is much higher than control groups,indicating that the HGF can accelerate the vascular growth evidently. We find that the weight of all these three groups of mice has no significant difference and it shows that the HGF can not affect the general growth of nude mice.The above results lead to the conclusion that 1,HGF gene can be expressed successfully in the adipose-derived stem cells and the generated HGF can form the effective density.2,HGF can be applied in the fat transplantation.It can improve the survival of implanted adipose tissue by accelerating the vascular forming in the implanted adipose tissues.This research provides the experimental base for the research on the application of HGF in transplantation of adipose tissue,effective methods and scientific basis for the revascularization research of adipose tissue engineering and also provide new ideal for the clinical application of transgenic tissue engineering.