Effects of mesenchymal stem cells transfected with human hepatocyte growth factor gene on healing of burn wounds.

OBJECTIVE

To explore the effects of bone marrow-derived mesenchymal stem cells (BMSCs) transfected with adenoviral vector carrying hepatocyte growth factor (HGF, Ad-HGF) on burn wound healing.

METHODS

BMSCs from male Wistar rats were separated and purified with Percoll separating medium by density gradient centrifugation and cultured with DMEM containing 20% fetal bovine serum (FBS). Then BMSCs were transfected with Ad-HGF at the optimal gene transduction efficiency of 100 multiplicity of infection (MOI). The efficiency of transfection and the expression of HGF in the suspension were detected by flow cytometry and enzyme linked immunosorbent assay (ELISA) respectively. Thirty-two female rats were subjected to 90 degree centigrade water for 12 seconds to induce a partial thickness skin burn. The animals were randomly divided into mesenchymal stem cells (MSCs) treatment group (Group A), Ad-HGF treatment group (Group B), Ad-HGF-modified MSCs treatment group (Group C) and saline control group (Group D). On days 3, 5, 7, 14 and 21 postburn, HE and Sirius red stain were performed to observe the burn wound healing and collagen content. The content of hydroxyproline in wounds was also detected. Transplanted cells and the expression of (sex-determining region Y) SRY gene were detected by in situ hybridization and polymerase chain reaction (PCR), while the expression of HGF in wound tissues was detected by ELISA.

RESULTS

The result of flow cytometry showed that the transfection efficiency was 86.41% at 100 MOI. Compared with the control group, the content of HGF in the supernatant after transfection increased time-dependently and peaked at 48 h, showing significant differences at 24 h, 48 h, 72 h and 96 h (P less than 0.01). Results of HE stain revealed that the range of re-epidermidalization in Group C was significantly larger than that in other groups in the first week. Three weeks postburn, the epidermis was significantly thicker in Group C than in other groups and the nails of dermis inserted into the derma of burn wounds. Sirius red stain showed that the content of collagen I in Group C was much less compared with that in other groups 21 days postburn. In situ hybridization revealed an expression of SRY gene in burned female rats, consistent with the finding of PCR. Immunohistochemistry demonstrated the largest increase of HGF expression in Group C, whose contents of hydroxyproline, however, decreased on day 7 postburn. Compared with other groups, the content of HGF in the wounds of Group C increased obviously on day 14 after transfection (P less than 0.05) and there was no significant difference among Groups A, B and D.

CONCLUSION

Our study suggests that transplantation of MSCs modified with Ad-HGF has positive effects on the healing of burn wounds probably through differentiation and release of relevant cytokines.