Lentiviral Vector Carrying Green Fluorescent Protein Gene Transfection Experimental Study Of Several Important Cells Of The Skin Structure

1.Objective:Gene therapy has been involved in many areas of plastic and reconstructive surgery,such as wound healing,reconstructive flap preservation,tissue engineering,and so on.The key point of gene therapy is to choose a proper vector,by which ectogenous gene can be well imported, lead to an efficient,specific and stable expression.Viral vectors are widely used for their high transfection efficiency,among which the lentiviral vector seems to be the most ideal one.Its advantages include:1) both dividing and non-dividing cells can be transfected,2)high transfection efficiency in many cell types,3) a piece of large gene can be inseted,4) transgene is integrated into host chromosome,leads to a long time expression.Up to now there are few studies about gene therapy using lentiviral vector to tansfect cells related to plastic and reconstructive surgery.Considering the particular sense of skin in plastic and reconstructive surgery,this study used lentiviral vector to transfect some types of skin cells.The main purpose to find out the appropriate condition for lentiviral vector's tansfection,the precise transfection efficiency and whether or not it has an influence on cells' survival and growth.2.Methods:Fibroblasts,adipocyte precursor cells and vascular endothelial cells were cultured,and used in the following experiments:1)Using LV-eGFP with MOI=10,20,50,100 to transfect cells,then observe the GFP expression in a fluorescence microscope during one week.2)Detecting the transfection efficiency of LV-eGFP with a proper MOI by fluorescence-activated cell sorting.3) Comparing the number of living cells between transfected cells and non-transfected cells by MTT and trypan blue tests.3.Results:1) Fluorescence microscopic observation:a) The GFP expressed better as the MOI increased.The groups with MOI=20 showed a satisfactory GFP expression.b) After four days of transfection,GFP expression became significant,and showed no reduction in one week.c) With the same MOI,the groups added polybrene showed a better GFP-expression.2) FACS results:The one-week-transfection efficiency is larger than 50% in all cell types.After three weeks the transfection efficiency reduced about 30%contrast to one-week's data.3) MTT and trypan blue tests' results showed no difference between transfected groups and control groups.4.Conclusions:An appropriate condition for lentiviral vector to transfect has been learned.For the three cell types in this experiment,MOI=20 is enough relatively.The target gene's expression can be well detected afer four days,with a transfection efficiency larger than 50%,and last for a long time(at least three weeks).Besides,lentiviral vector's transfection has no influence on cells' survival and growth.