| Objective: A series of biological activities are involved in the process of wound healing after all kinds of injuries, which the expression of nucleus proteins, which is mediated by the transduction of cellular signals through MAPKs pathways, is the basis of all biological activities. In the present study, we studied the activation of cellular signal pathways in order to explore the rules of signaling pathways involved in the wound healing process. Meanwhile, the effects of exogenous bFGF on signal pathways were also investigated. Therefore, the objective of this study was to provide to certain extent the theoretical basis for the regulation mechanism signal network in the process of wound healing, appraise the effect of topical application of growth factors in clinic, so as to improve the quality of wound healing.Methods: Rats with partial thickness scald were used as animal model. The activities of signal pathways during the proliferation, migration, transduction and apoptosis of cells involved in the healing process were investigated, and the effects of exogenous bFGF (basic fibroblast growth factor) on the expression or inhibition of signal proteins were observed. Apoptosis of fibroblast in vitro was induced by heat-stress. Mitogen-activated protein kinases (MAPKs) pathways were activated or inhibited to observe the expression of downstream proteins. Skins from fetus, adults and elderlies, and tissue specimens with different healing outcomes (including hypertrophic scars, chronic ulcers and normal scars) were included in the study. The expression changes in receptor tyrosine kinase at different developing stages were examined. The methods adopted were: immnohistochemistry, immunofluorescence, in situ hybridization, DNA gel electrophoresis, transmission electron microscope, routine HE and specific V.G staining, RT-PCR, confocal microscopy, and Western blotting. The expression of proliferation and apoptosis related genes including caspase, p53, Bcl-2, Bax, c-myc were also determined. Different expressions of EGF (epidermal growth factor) and bFGF and their receptors were compared.Results: The results were summarized in three sections as following:1. The effects of exogenous bFGF on proliferation, apoptosis, transformation, re-epithelization and migration of cells after scald injuryExogenous bFGF induced the expression of c-myc in fibroblasts in wound sites, and resulted in the activation of p53 proteins, which subsequently mediated the changes in PCNA and caspase 3, MAPKs participated in this process. Exogenous bFGF also increased the expression of TGF-pl, promoted the conversion of fibroblasts to myofibroblasts. There was no significant effect of exogenous bFGF on proliferation of vessel endothelial cells. However, it did evidently change the expression of FAK. At the same time, the phosphorylation of ERKs was promoted obviously, indicating that MAPKs might mediate the migration of vessel endothelial cells through ERK pathway. After the administration of bFGF, the phosphorylation of MAPKs around wound edge and in deep skin appendages were enhanced, accompanied by active expression of pi integrin, E-cadherin and FAK. bFGF promoted the expression of matrix metalloproteinases in wounded areas, increased the migration of keratinocytes, regulated the compositions of extracellular matrix, and kept the balance of synthesis and degradation of collagens.2. Thermal injury induced cell apoptosis and growth factors involved in the regulation of signal networksFibroblasts apoptosis was induced by lOmin incubation at 45 °C water bath in DMEM medium with 5% fetal bovine serum (FBS). Changes in MAPKs signal pathways (Erkl/2 and INK) were observed after MAPKs inhibitors and bFGF was added to thermal injured fibroblasts cultured in vitro. There was interaction between two pathways. Exogenous bFGF could induce phosphorylation of MAPKs and vibration of intracellular calcium. After the addition of MAPKs inhibors PK98059 and SB203580, the expression of intracellular calcium was inhibited. Exogenous bFGF stimulated the activaton of STAT1 and ERK 1/2 signaling pathways. After the addition of MAPKs inhibors PK98059 and SB203580, the expression of STAT1 was affected. The interactions among pathways mediated the expression of c-fos and c-myc, resulting in either apoptosis or proliferation.3. Effects of receptor tyrosine kinaseThe expressions of EGFR and bFGFR were different between normal skin and hypertrophic scar. The expression levels in hypertrophic scars were singificantly higher than those in normal skins.The expression of EGFR and FGFR were distributed in whole epidemiis, while there was very little in the dermis of fetus. The expression of both receptors was increased with the increase of gestational age. The expression of EGFR was mainly localized in the basal layer of keratinocytes in postnatal skins. Both of them were decreased in membranes of all kinds of cells in aging skins.Conclusions1. Exogenous bFGF mediated the proliferation of fibroblasts and keratinocytes, the migration of vessel endothelial cells, and affected apoptosis of cells through the activation of transcription factors c-fos and c-myc through ERKs signal pathway.2. Exogenous bFGF could promote the secretion of other growth factors, collaborating in converting fibroblasts to myofibroblasts, and finally affecting the outcome of wound healing.3. The expressions of pi integrin and E-cadherin were important markers of epithelial migration. The beneficial effects of exogenous bFGF on wound healing process might be associated with activities of stem cells located in hair follicales and basal layed of the epidermis. The increase of phosphorylation of MAPKs might regulate the repair acitivites of epidermal stem cells.4. The expression of MMP-1/2 and TIMPl/2 had close relationships with the activation of ERK1/2 signal pathway during the wound healing process. Oncogene c-fos mediated the regulation process.5. Thermal injury could induce apoptosis of fibroblasts in vitro. Erkl/2 and JNK were important pathways in initiating apoptosis. There were interactions among MAPKs subgroups. There was a feedback regulation mechanism between Ca2+ and MAPKs pathways. MAPKs and STATs pathways had cross-talking.6. Different expression of receptor tyrosine kinase was one of the major factors producing different outcomes of wound healing. |
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