| protein injection and gene transfection. Because VEGF protein is very expensive and its half-life is very short and easy to be degraded, also because large dose of VEGF could cause changes in blood dynamics, the application of VEGF protein injection in clinic has been restricted. With the development of gene transfection techniques, the transgenic therapy of VEGF has become an effective way in exerting its function locally. Previous studies have reported the application of VEGF transgenic therapy in ischemic diseases, e.g. limb ischemia, cardioischemia and skin clap survival, few studies have focused its usefulness on CW healing.The present study was designed based on above background. A modified rabbit ear ischemic wound model, the nanosphere complex-mediated VEGF plasmid and gene transfection technique were used to investigate the role of VEGF in promoting CW healing and the underlying mechanisms.Part I: Establishment of CW model.Healthy New Zealand white rabbits (n=8) were divided into two groups, aged group (48-60 months old, n=4) and young group (3-6 months old, n=4). Cyclic cut was conducted at the ear roots of rabbits, the central artery and cephalic artery were separated and ligated, The caudal artery and all veins were remained. Then three circular wounds (diameter=6 mm) were constructed at ventral ears with biopsy punch. Scraped the perichondrium to expose cartilage. At the third, 6th, 9th and 12th day after operation, cyclic cut was conducted at the ear root of rabbits of aged group respectively and blocked blood flow to the maximum. Observed and determined the skin temperature of rabbit ear, oxygen tension of blood in vein, and new epithelia gap (EG), new granulation tissue gap (GTG) and the peak height (PH) of new granulation tissue under microscope. The result showed: (1) that the skin temperature and oxygen tension were significantly different after surgery with that before surgery in both groups (P < 0.01). Significant difference was also existed between aged and young groups at each time point 3 days after surgery (P < 0.01). The skin temperature and oxygen tension in aged group was significantly slower than in young group (P<0.01). (2) The determination of HE staining showed that EG and GTG in the old-aged group increased 72% and 67 %, respectively, when compared with that in young groups (P < 0.01). While, the PH in aged group only decreased 6% (P>.05).Part II: Construction of nanosphere-mediated human VEGF expression vector.This part of experiment was to construct nanosphere-mediated VEGF expression vector and compared the transfection efficiency with lipofection transfection method. Human VEGF165 DNA fragment was used as target gene and recombinant expression vector pcDNA3.1/myc-HisA-VEGFi65 was constructed. Nanosphere and lipfection methods were used to mediate the tranfection of VEGF165 into mouse fibroblast cells (3Y1). The changes in cell phenotype was observed under microscope; The expression of green fluorescent protein was used to observe the transient transfection rate; After screened by G418, the expression of VEGF in the culture supernatant wasdetected by ELISA; Western bolt was used to examine the expression of gene products; MTT method was performed to detect the toxicity to cells. The results showed that the positive clones screened by G418 were mouse fibroblast cells with high expression level of VEGF. Immunohistochemistry revealed that recombinant pcDNA3.1/myc-HisA-VEGFi65 was highly expressed in transfected cells. Western blot detected the expression of VEGF in cells and supernatant 48 hours after transfection with nanosphere, reached a peak at 3-5 dyas. Immunofluorescent method indicated that the transfcetion rate of nanosphere was significantly higher than that of lipofectin.Part III: Effects of VEGF transfection on CW healing.New Zealand aged rabbits (n= 8) were used to generate CW models. A non-ischemic wound with similar size in each ear was also produced as a control. Animals were divided into three groups: CW+VEGF group (add nanosphere-mediated VEGF onto C...
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