| Objective To investigate the effect of 12-Alkylated Chitosans / Plasmid-encoding Vascular endothelial growth factor(VEGF) gene transfection on early pedicle division of random flap in rats, and compare with plasmid-encoding VEGF.Methods The experiment was accomplished in the Molecular organism Laboratory of Anhui Medical University and the Surgical Experimental Animal Center of the First Affiliated Hospital from March to October, 2008. 12-Alkylated Chitosans / plasmid-encoding gene were prepared by using eukaryotic expression plasmid pcDNA3.1-VEGF121 that have been constructed by gene engineering. Then check the DNA protection of gene in vitro; Forty male healthy Sprague-Dawley rats were randomly divided into four groups: destination group, Plasmid-encoding VEGF121 group (Plasmid group), 12-Alkylated Chitosans group(CS group) and Blank control group with 10 rats in each one. The random flap(2cm×6cm) in full-thickness with pedicle at the iliac crest's lever was designed on all of the experimental rats. After the skin flap formed, design 10 injection-sites in each rat. Four various reagents were respectively injected into the skin flap in the Destination group, Plasmid group, CS group and Blank control group immediately at the operation (100ul per-site and 1ml total in each one). As soon as administration, 3-0 suture silk was used to conduct suturation in situ. Antibiotics was used to infection prevention, and then rats were raised separately. The pedicles of all rats were divided 4 days later. On the 7th day after pedicle division, the survival rate of all skin flaps were investigated, and skin flap specimen were stained with hematoxylin and eosin whose histological changes were observed under light microscope. The expression of VEGF121 was detected with immunohistochemical stain, and the density of capillary was calculated by CD34 immunohistochemical stain.Results The first, eukaryotic expression plasmid pcDNA3.1-VEGF121 was extracted with alkaline lysis successfully. After confirmed with enzyme re-digestion, pcDNA3.1-VEGF121 was used to preparing 12-Alkylated Chitosans/Plasmid-encoding VEGF121 Complex Nanoparticles (12-ACS/pcDNA3.1-VEGF121) with 12-ACS.The nanoparticles have been proved the complete of plasmid embedding and the DNA protection of gene. The second, on the 7th days after pedicle division, all the experimental animals were survived. The limits between survival and necrotic was identified. The differences in survival rate and density of capillary sections in flaps were remarkable between the destination group and control group(sP<0.01). Survival rates of destination group, Plasmid group , CS group and Blank control group were (85.40±3.22)%, (71.27±3.48)%, (50.45±4.69)%, (49.23±3.04)% ; The density of capillary sections in above four groups were 43.15±2.33 /HP, 33.17±2.76/HP, 23.27±2.21/HP, 30.63±8.71/HP respectively. There was marked differences not only between Plasmid group and CS group, but also between Plasmid group and Blank control group(P<0.01). But there were no marked differences between CS group and Blank control group(P>0.05). Immunohistochemical staining proved that buffy granula staining can be found in all groups which were the most and deepest staining in the destination group. The fewest group was Blank control group. Through histological observation, there were abundant blood capillaries and granulation tissue can be found in the destination group, while those in the Plasmid group were less. Although it is better than CS and Blank control group.Conclusion 12-ACS can be safely used as genetic carrier during the transfection of VEGF121 in rats'skin flap. The efficiency of transfection and gene expression were higher than pcDNA3.1-VEGF121. Localised application of VEGF121 can ameliorate the microcirculation and accelerate vascularization of the flap which change the condition of blood supply in the distal flap. 12-ACS/pcDNA3.1-VEGF121 can also help the flap survival and improve earlier division of random flaps. |
