Effects Of Mesenchymal Stem Cells With Transfection Of Human Hepatocyte Growth Factor Gene On The Healing Of Burn Wound After Heterogenous Grafting

Objective 1. To develop a gene agent which active cytockines could be expressed effectively.2. To explore the effects of bone marrow-derived mesenchymal stem cells (MSCs) with transfection of adenoviral vector mediated human hepatocyte growth factor (Ad-HGF) on the healing of burn wound.Methods part one MSCs of male Wistar rats were collected and cultured, and the expression of C—met were detected by immunohistochemical examination. MSCs were transfected with Ad-GFP in different MOI in order to select an optimal MOI. Transfection efficiency was assayed by fluorescent microscopy and flow cytometry. The expression of HGF in MSCs transfected with optimal MOI was measured with ELISA assay. MTT method was used to evaluate the proliferation of MSCs.part two 50 animals were subjected to 90°C water for 5 seconds to produce a partial-thickness skin burn. Animals were randomly divided into 5 groups: MSCs treatment group (group A); transplantation group of Ad-HGF modified MSCs (group B); transplantation group of Ad-GFP modified MSCs (group C);Ad-HGF treatment group(group D); saline control (group E). Suspension of MSCs, suspension of Ad-HGF and saline were injected into the derma of burn wounds after injury. 3, 5, 7, 14, and 21 days postburn, histological examination including HE and VG stain and contraction rate of burn wounds were performed to evaluate wound healing. At the same time, analysis of hydroxyproline content in wound tissue were done. In situ hybridization was used to detect the existence of the transplanted cells. Immunohistochemical examination was carried out to detect the positive expression of HGF gene in group B.Result 1. The expression of C -met gene was detected on cell plasm and membrane.2. The highest transfection efficiency could be achieved when MOI equaled 100.3. The expression of HGF in MSCs reached at the highest level after being transfected with Ad-HGF for 48h.4. The proliferation of MSCs was prohibited when they were transfected at 200 of MOI.5. During the first and second week after burn, the contraction rate of burn wound in group B was much higher than that in group A, C, D and E .6. Histological examination showed the thickness of epidermis in group B was significantly thicker than that in the other groups, the nails of dermis stretched into the derma of burn wounds.7. Hydroxyproline contents of wound tissues in group B was significant different when compared with other groups , especially 7 days after burn.8. The arrangement of collagen in group B tissue appeared more orderly.9. The expression of male Sry gene was watched in the burn wound of female rats in situ hybridization.10. Immunohistochemical examination demonstrated that HGF expression increased significantly in group B compared with other groups.Conclusions1. MSCs were ideal cells for cell-mediated HGF gene therapy or stem therapy.2. With the increase of Adenovirus, the proliferation of MSCs could be restrained.3. Transplantation of MSCs with Ad-HGF transfection had more positive effects on the healing of burn wounds than transplantation of MSCs or injection of Ad-HGF solely, which may open a new way for burn wound repair.