| Cutaneous wound healing is a complex process involving a series of sequential and overlapping phases, including inflammation, formation of granulation tissue, deposition of new extracellular matrix (ECM), and remodeling. Each of these stages is dependent upon responses of resident fibroblasts that include proliferation, synthesis of ECM, and migration. In previous study has confirmed SPC could accelerate skin wound healing. Its mechanism may be related to SPC stimulated the proliferation of several cell types in vivo, such as keratinocytes, fibroblasts, cells around sebaceous glands and hair follicles as well as endothelial cells, and improved neo-angiogenesis, and accelerate cell migration, adhesion, cytoskeleton rearrangement and increase various of protein expression associated with cutaneous tissue, it can be combined a specifically of cell surface receptor and then activated signal transduction pathway, stimulation of cytokines activities, and as a second messenger roles in a various cell signaling pathways, play a role in skin wound healing, but until now has not been clearly.Tissue remodeling relies on extracellular matrix (ECM) restructuring by the matrix metallopeptidases(MMPs), which is also controlled by specific tissue inhibitors of metalloprotein-ases(TIMPs). MMPs and TIMPs family play key roles in maintaining the balance between ECM deposition and degradation. TIMPs are acknowledged to play other roles in human physiology and pathology in addition to MMP-inhibition, regulating proliferation and survival of various normal and tumour cells and this should be recognized also when clarifying regulatory mechanisms of wound healing. Therefore, by understanding the variation of the TIMP-1 expression can be from one side to evaluate the efficiency in wound healing.Objective:This study was purposed to investigate the effects of the expression of TIMP-1 gene induced by SPC in human dermal fibroblast cells and could be a hopeful knowledge that the role of SPC accelerated skin wound healing process.Methods:Fibroblasts isolated from foreskin obtained during circumcision 3 cases were cultured in monolayer, and the experimental study of the fibroblasts at less than passage 12. The human dermal fibroblasts were cultured in media with different concentrations of SPC, was added with SPC of the concentrations at the concentration of 0.1,1,5, 10,20μM respectively contrast to vehicle and co-cultured for several hours of SPC 5μM. The amount of fibroblast proliferation was assessed using MTT assay. The SPC-induced TIMP-I mRNA in human fibroblast was measured by reverse transcription and polymerase chain reaction (RT-PCR).The presence of SPC-induced TIMP-1 protein was confirmed by Western blotting analysis.Results:Administration of SPC results in a dramatic increase in proliferation of human dermal fibroblast, it was significantly higher in the 5μM and lOμM experimental group than in the control group (P<0.01), it showed that SPC increased proliferation of human dermal fibroblast in a dose-dependent manner. RT-PCR showed that 5μM SPC resulted in stimulation of TIMP-1 gene expression and SPC-induced stimulation in a concentration-dependent manner. When the concentrations of SPC were 10μM and 20μM, the expression of TIMP-1 gene was decreased. After the human fibroblast were treated with 5.0μM SPC for 2,6,12,24, and 48 hours, the expression of TIMP1 mRNA was increased to various degree. This increased effect displayed time-dependent manner and the most optimal effective time was 6-12 hours (p<0.05). Western blotting analysis showed that SPC treatment increased the level of TIMP-1 protein, in a time-dependent manner (p<0.01).Conclusion:This observation suggested the possibility that SPC regulate TIMP-1 gene expression, and may be involved in wound healing process. |
