The Experimental Study On MSCs Transfected By EGF Gene For Wounding Healing

Objective:Refractory wounds following skin tissue defect are mainly caused by the lack of repair cells and the reduce of biological activity facors involved in would healing process. In the absence of sufficient autologous skin, it remains a difficulty for clinic treatment to decide how to restore effective wound repair and obtain the physiological healing of the skin timely and effectively. Therefore, we try to modify bone marrow MSCs with recombinant AAV (adeno-associated virus)-mediated EGF gene as a way to explore its potential for promoting wound healing, which may provide some theoretical and practical basis for the source of cells for skin tissue engineering and transgenic technology in wound healing.Methods and Results:1. Construction of pAAV-EGF-GFPIn this study, we amplified the 175bp fragment of EGF gene from the placenta by RT-PCR and cloned the cDNA into vectors. The pMD18-T-EGF vectors were then digested with EcoRI and SaiI and cloned into the multiple cloning site of plasmid AAV-IRES-GFP. And the successful construction of pAAV-EGF-GFP was identified by restriction enzyme and sequencing analysis.2. The packaging and purification of AAV-EGFThe calcium phosphate precipitate is formed by mixing pAAV-RC, pHelper and pAAV-EGF-GFP. The recombinant AAV-EGF was purified and condensed as the method including chloroform treatment, PEG/NaCl preciptation and chloroform extraction. The etiterof AAV was was detected by SDS-PAGE, and the purity by DNA dot-blot.3. Isolation and culture of bone merrow mesenchymal stem cells Mouse bone marrow-derived MSCs were isolated and purified by dsnsity gradient centrifugation and direct adherence separately. The MSCs cells were induced to osteoblasts, chondroblasts and adipocytes, identified by Von Kossa, Alcian Ponceau and oil red O staining methods. The results showed that MSCs that had multiple differentiation potentials could be obtained by primary and passage culture.4. Transduction of MSCs with recombinant AAV-EGF3d,14d,27d post-transduction, the total RNA were extracted from the transduced MSCs and detected by RT-PCR method using the specific primer for EFG gene. The results showed that AAV-EGF vectors successfully transfected MSCs and mediated the expression of EGF gene in vitro.ConclusionMSCs that have the proliferative activity and potential to differentiate were successfully isolated and cultured in vitro. EGF cDNA was cloned into plasmid pAAV-EGF-GFP. Recombinant AAV-EGF vectors could mediate the expression of EGF gene in MSCs in vitro.