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With the development and progress of society, increasing concerns about the systemic and local ionization damage and these ionization injury from: nuclear accident, radiation cancer treatment, long X-ray, nuclear medicine overdose radioactive inhalation of materials, nuclear explosive terrorist incidents. Body injury after coordination of the healing process, including a variety of cells, cytokines, and extracellular matrix between the intricate network role. Normal wound healing paracrine or autocrine cytokine or growth factor-mediated regulation cascade amplification reaction realized. Put invasive healing process combined injury impaired wound healing disorders, repair cells reduce the number or function is impaired, resulting in the growth factor plays an important role in wound healing synthesis decreased collagen synthesis and decrease in inflammatory cells. The functional characteristics of mesenchymal stem cells in the bone marrow (bone marrow-derived mesenchymal stem cells, BMSCs) to promote wound healing include: able to migrate into the site of trauma or inflammation involved in the regeneration and reconstruction of damaged tissue, stimulate the inherent progenitor cell proliferation and differentiation in different directions, through the secretion of growth factors and to reshape cells to promote the recovery of damaged cells, play a special immunomodulatory and anti-inflammatory effects. Effective way to BMSCs as a repair cells and / or exogenous gene expression vectors used in clinical is currently a hot research topic. Function of the vascular endothelial growth factor (vascular endothelial growth factor, VEGF) as promoting endothelial cell division agent chemotaxis medium and vascular permeability inducers, it is only in the wound healing cascade from the multi-functional role of the factors, including angiogenesis, endothelial formation and collagen deposition. Local microbial persistent infection of wounds the refractory or one of the reasons unhealed. Defensin is rich in positive charge, acts on the cell membrane, having a broad spectrum bactericidal activity. Because of its unique mechanism of action, almost without resistance, in which the human β-defensin-3 (human beta defensins hBD-3) has played an important role in the prevention and treatment of infectious diseases in humans, in a small dose range showed antibacterial activity against Gram-positive bacteria, the antibacterial activity of non-salt concentration sensitivity, nor cause hemolysis, small molecular weight, synthetic, fast diffusion does not harm human tissue cells, is an ideal anti-infective activity antimicrobial peptides. Studies have shown no difference hBD-3 recombinant and chemically synthesized hBD-3 antibacterial properties and biological properties with the separation out of the natural peptide. In order to study and promote the healing of chronic skin ulcers injury put record combined injury, we build pVIVO1-hVEGF 165 -hBD-3 eukaryotic plasmid vectors for the transfection of rat BMSCs and hVEGF 165 / hBD-3 gene-modified BMSCs transplanted to the wound subcutaneous the rats put record combined injury to the synthesis and release hVEGF 165 of hBD-3 jointly promote wound healing. The main methods and results are as follows: 1.hVEGF 165 / of hBD-3 double gene co-expression of Construction and identification of eukaryotic plasmid vector. Amplified by RT-PCR method from leukemia cells (HL60) and gum tissue hVEGF 165 and hBD-3 gene fragments by recombinant DNA technology, gene fragment has restructured in pVIVO1-mcs eukaryotic expression vector to construct the recombinant plasmid vector pVIVO1 hVEGF 165 and pVIVO1-hVEGF 165 -hBD-3 enzyme electrophoresis analysis and DNA sequencing methods reorganization The plasmid vector identified. The transfection reagents recombinant plasmid transfected BMSCs use jetPEI RT-PCR, immunofluorescence cytochemistry and Western blot confirmed recombinant eukaryotic plasmid transfected BMSCs successful high expression of of the exogenous gene hVEGF 165 of hBD-3 2. the the recombinant plasmid pVIVO1-hVEGF 165 -of hBD-3 transfected BMSCs expression product activity identification. Collected separately transfected the recombinant the plasmid pVIVO1-hVEGF 165 -hBD-3 (T), the recombinant plasmid transfection pVIVO1-hVEGF 165 (C1), transfected with empty vector (C2 ) and normal (N) of untransfected BMSCs serum-free culture supernatant, were named T-CM, C1-CM, C2-CM and N-CM. To detect secreted by the hVEGF 165 can stimulate cell proliferation and migration, Another established group T-CM/Ab, namely T-CM hVEGF 165 and antibodies to blocking hVEGF 165 action effects. Good state of growth of endothelial cells EA.hy926 4 × 10 3 / well were seeded in 96-well plates containing 100μL / well of complete medium. The conventional culture after 24 hours, each hole to replace the above-mentioned five kinds of conditioned medium, 5d proliferative effect in order to observe continuously detected according to the CCK-8 kit instructions. The results showed that: compared with C2-CM, N-CM and T-CM/Ab, T-CM and C1-CM group in the 1-4d can significantly promote cell proliferation (p lt; 0.05), indicating that the expression of exogenous gene hVEGF 165 promote cell proliferation. When endothelial cells the EA.hy926 inoculated on 12-well plates and wait until it reaches 95% of fusion, using a sterile 1mL pipetting head, in the central vertical cross-culture plate bore to draw a \scratches, and then washed with PBS 2 times, then 1 mL of the above five kinds of conditioned medium was added to each hole. 0, 4, 8, 12 and 18 h after the scratch using an inverted microscope at the \Image analysis software to measure the healing rate. The results showed that the T-CM and C1-CM group scratches after 4, 8, 12 and 18h ??healing rate was significantly higher than the C2-CM, N-CM and T-CM/Ab groups (p lt; 0.05), indicating that Expression of exogenous gene hVEGF 165 the role of promoting cell migration. Above four culture supernatant freeze-dried, reconstituted, and access to a 20-fold concentrate, then by KB observation of Escherichia coli (EC, ATCC 25922), Pseudomonas aeruginosa (PA, ATCC27853), Staphylococcus The antimicrobial effect of Staphylococcus aureus (SA, ATCC25923), Bacillus subtilis (BS, ATCC62037) and Candida albicans (fungi, CA, ATCC10231). The results show that, T group culture the supernatant concentrate significant inhibition zone, other groups no inhibition zone. Show that the recombinant plasmid after transfection BMSCs, expressing exogenous gene hBD-3 suppressed the role of bacteria and fungi. 3. Recombinant the plasmid pVIVO1-hVEGF to 165 -local transplantation of hBD-3 transfected BMSCs put record combined injury wound repair. Full-thickness skin wounds replication rats put record combined injury, injection of local punctate transplant 165 -hBD-3 of BMSCs transfected with the recombinant the plasmid pVIVO1-hVEGF T group transfected with recombinant injection transplant The BMSCs the plasmid pVIVO1-hVEGF 165 for the group C, injection transplant normal untransfected BMSCs group N, S group injected with PBS. 1d, 3d, 7d, 13d and 20d after transplantation, the general observation wound healing, simultaneous determination of residual area and healing time. Were taken at each time point paraffin sections were stained with HE, observed in each group at each time point wound pathological changes; paraffin sections after Sirius red staining, polarized light microscopy observation of changes in the collagen fiber content; immunohistochemical staining The exogenous gene hVEGF 165 / of hBD-3 and the inner layer of granulation tissue laminin (Laminin) expression. The results show that the general observation at each time point, compared with the C, N, S group, to a lesser extent T wound infection, faster healing, the average wound healing time of 20d, while the C, N, S, the group's average The healing time is 22-23,24-25 and 27-28d. After transplantation 7d, 13d and 20d, T group remaining residual area percentage was significantly lower than the other three groups (P lt; 0.05); HE staining results confirmed the T group better granulation tissue formation; Sirius red staining of collagen fibers results the T group collagen fiber content and the thickness was significantly higher than the other three groups; wound immunohistochemistry observed T exogenous gene hVEGF 165 / hBD-3 expression of, and wound granulation tissue reflected Laminin expression of vascular basement membrane formation was strongly positive. T and C groups, the differences can be speculated: of hBD-3 in wound healing may play a role, perhaps with the antimicrobial activity of hBD-3. Differences in group C and group N can be speculated: hVEGF 165 inherent promote endothelial cell proliferation and vascular characteristics may play in the process of wound healing role. The local transplantation genetically modified BMSCs really can promote wound healing, which is probably due to the secretion promote endothelial cell proliferation, angiogenesis hVEGF 165 and antimicrobial of hBD-3, was a result of wounds at more good granulation tissue formation / maturation, the skin attachment remodeling and a lower rate of infection. These data confirm that this stem cell therapy and gene therapy jointly applied to impaired wound healing potential.
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