Experimental Study On Adipose-derived Stem Cells Transfected By Recombined Adenovirus Vector Carrying VEGF165 Gene

This study is to isolate human Adipose drived stem cells and regard them as target cells of gene therapy,construct Adenovirus vector carrying human vascular endothelial growth factor cDNA.The purpose is to combine ADSCs and VEGF₁₆₅by gene transfection and explore an effective method for adipos tissue engineering。Objective:1.To investigate the biological characteristics of mesenchymal stem cells from human adipose tissue;2.To construct recombined Adenovirus vector carryingVEGF165 gene;3.To establish the best condition of Ad₅ recombinant adenovirus vectors on infecting human adipose mesenchymal stem cells,which was related with MOI;4.To observe the efficiency of VEGF₁₆₅ expression after transfecting ADSCs by recombined Adenovirus vector carrying VEGF₁₆₅ gene;5.To observe the ability of infected cell proliferation and celluar cycle of ADSCs.MethodsHuman adipose mesenchymal stem cells were obtained by collagenase digestion method and cultured primarily and observed ADSCs'biological characteristics,The surface phenotype of cultured adipose mesenchymal stem cells was identified by flow cytometry and cell growing line was draw.2.Under the conditions of Multiplicity of Infection(MOI)in gradients,Ads recombinant adenovirus vectors infecting human ad- ipose mesenchymal stem cells were carried out in different time.Subsequently,the infection efficiency was assayed by fluorescent microscopy and image analysis.3.The experiments was divided into two groups:AD₅-VEGF₁₆₅ transfection group,and controll group.Experimental group was used Adenovirus vector carrying VEGF₁₆₅gene to transfect into the ADSCs and the transfection efficiency was evaluated by transfecting EGFP gene.The subsequent VEGF₁₆₅expression was detected by ELISA.4.MTT assay was used to evaluate cell proliferation and cellular activity.The cell circle was tested by flow cytometry analyze.Result:1.Adequate ADSCs were obtained by direct culture cell of adipose tissue and the cells were passaged on the 10th day,and flow cytometry tests showed that CD106 was neg- ative and CD49d was positive.2.The best infection rate could be reached when MOI was 400.The flow cytometric analysis showed the decrease of the multiplication and the increase the cells transfected with adenovirus.The recombinant adenovirus infe- ction rate was higher.when MOI was 400 pfu/cell,a comparatively ideal infection efficiency was achieved.3.ELISA assay showed that the expression of transefction group was significant difference compared with that in control group(P＜0.05).4.Compared with group which transfected with VEGF_165groupthat infected with adenovirus and controlled group,There is no obvious differences in ADSCs OD value,the three groups had same percentage of DNA G1period and PrI(P＞0.05).Conclusions1.There is abundant ADSCs in human adipose tissue,and their biological characteris- tics is stable.2.The recombinant adenovirus is an ideal vectors for human adipose mesenchymal stem cell.An appropriate MOI could improve the infection efficiency by means of promoting cells to intake AdMaxTM recombinant adenovirus.3.ADSCs can be infected with VEGF_165-gene.The infected ADSCs could express the target Protein effectively in vitro.The transfection of adenovirus vector doesn't effect celluar cycle of ADSCs.SoVEGF therapy and transplant of stem cells together by adenovirus vector and this method may play a new role in the treatment of ischemic.