Promotion Of The Healing Process Of Refractory Wound By HVEGF165 Gene Transfected With DMSCs

Combined injuries of wound and radiation often occur in nuclear accidents and attacks. This kind of wound usually has a common rule: prolonged wound healing process is its primary character. Though there are many approaches to speed up the process of wound healing, such as dressing, medicines, local application of cytokines and skin graft, etc, there is no approach that really works long. Facing these wounds, we have to explore new approaches to promote refractory wound healing.Recently, with the rapid development of cell biology and molecular biology, the process of study on mechanism of refractory wound indicated there are several reasons responsible for the prolonged healing process of refractory wound: one is its poor local circulation, another is the depletion and lack of local repair cells; and still another one is that there are short of or decreased cytokines in wound environment. The development of stem cell engineering and transgenic method in our Institute showed that transgenic cell transplantation not only provides enough repair seed cell but also the high expression of the target gene will make up or resolve the problem of local cytokines. Dermal multipotential stem cells (DMSCs), a kind of cells separated from the mesoderm of fetal rat, not only have high proliferation ability but also are capable of multi-differentiation into osteoblasts, chondrocytes, fibroblasts etc. Research results showed us DMSCs play an important role in wound healing, their responses to wound circumstance are much more stronger than that of fibroblasts, vascular endothelial cells and other repair cells. Moreover, they are easy to be cultured and proliferated in vitro, and theoretically ,can keep its multi-differentiating potential after many passages. In this study, the results indicated DMSCs are easily infected by plasmid gene carrier and efficiently express the target gene. Therefore, DMSCs can be regarded as an ideal seed cell in constructing engineering skin to treat big area skin wound and a suitable target cell for extrogenic gene expression and cellular gene therapy. VEGF165, an isoform of vascular endothelial growth factor family A, has the strongest biological function in promoting the form and growth of blood vessel. As a kind of secretory glycoprotein with strong ability of diffusion, it can easily reach the target cells and plays an important part in promoting the migration, proliferation, secretion and survival of vascular endothelial cells, fibroblasts and other cells. Thus, it plays a powerful role in promoting wound healing.Based on the reasons mentioned above, we make use of the potential of DMSCs ' self-renewing and multi-differentiation, and transfected a bicistron eukaryotic expression vector, pIRES2-EGFP-hVEGF165, labeled with enhanced green fluorescent protein (EGFP) into DMSCs. After in vitro culture, the transfected DMSCs were grafted into the wound area in animal model to explore a new way to promote refractory wound healing.The main results are as follows.1.Eukaryotic expression bicistron plasmid vector pIRES2-EGFP-hVEGF165 was constructed successfully. After the total RNA of HL-60 were harvested, we use specific primer of hVEGF165 and hVEGF165 cDNA was obtained by using reverse transcription polymerase chain reaction technology (RT-PCR). Purified with electrophoresis, the objective fragment was ligated with pMD18-T vector in the presence of T4 DNA Ligase. The obtained pMD18-T vector-hVEGF165 was then transferred into an engineered bacterium DH5α to replicate the target plasmid. After the identification of inserted hVEGF165 fragment with restriction endonuclease EcoRⅠand BamHⅠdigestion, the DNA sequence of hVEGF165 was confirmed by using DNA sequencing device. The fragment sequence was confirmed to be consistent with the reported one in Genbank. Then those plasmid vectors with correct hVEGF165 flagment were harvested. After the hVEGF165 cDNA fragment was cut from those very pMD18-T vector -hVEGF165 with EcoRⅠand BamHⅠand purified with electrophoresis, it was inserted into the m...