The Research About The Expression Of Protease-activated Receptor-2 On Autologous Skin Graft With Hyperpigmentation

BackgroundAutologous skin grafting is normal operation in Plastic and Reconstructive Surgery,but the recipient site become hyperpigmented may be an annoying problem.the main reason is due to the accumulation of melanin granules in the epidermal of the skin grafts,especial on face and joint.It affects mental healthy and socialty function.The mechanism is not clear,and it’s difficult to resolve.ObjectiveDetecting the amount of melanin and the expression of protease-activated receptor-2 in autologous full-thickness skin graft with hyperpigmentation and detecting the amount of melanin change of hyperpigmented skin grafts after treated with soybean trypsin inhibitor to discuss the protease-activated receptor-2 function of in autologous full-thickness skin grafts with hyperpigmentation and look for a possible mechanism of hyperpigmentation after autologous full-thickness skin graftingMethodDevide 30 guinea pigs to A、B、C groups,treated with autologous full-thickness skin grafting, observing the processing of hyperpigmentation to selected the ideal model of autologous full-thickness skin grafing with hyperpigmentation from 2nd to 16th week.Using Masson Fontana stain to observe the mount of melanin and distribution. Using immunohistochemistry, western-blotting, real-time polymerase chain reaction to observe the expression of protease-activated receptor-2 between the hyperpigmented skin graft and control normal skin.Take the autologous full-thickness skin grafts with hyperpigmentation black guinea pigs divided into two groups:the right skin grafts settled to A group; the left skin grafts settled to B group.the A group is experimental; the B group is controlled.A group treated with the protease-activated receptor-2 activation inhibitor soybean trypsin inhibitor(50μM) three times a day,7 days a week, for 4 weeks.Group B were treated with placebo(ethanol:propylene glycol mixture,70:30, v/v).Using Masson-Fontana stain and image pro plus 6.0 soft to observe and analysis the change of the amount of melanin in skin grafts.Result1. Using Masson-Fontana stain and soft image pro-plus6.0 analysis to find the autologous full-thickness skin grafts melanin MOD of white guinea pigs have no statistically significant differences from 2nd to 16th week (P> 0.05); autologous full-thickness skin grafts melanin MOD of brown guinea pigs have statistically significant differences from 2nd to 3rd week(P< 0.05),but have no statistically significant differences vs control from 4rd tol6th week(P> 0.05); autologous full-thickness skin grafts melanin MOD of black guinea pigs have statistically significant differences vs control from 2nd to 4th week,as well as have statistically significant differences particularly at 16th week(P< 0.05),but have no statistically significant differences from 8rd to 12th week(P> 0.05).Treated with protease activated receptor-2 activation inhibitor STI with 50μM on autologous full-thickness skin graft with hyperpigmentation after 4 weeks, the melanin IOD of skin grafts were statistically significant difference (P< 0.05)vs treated with placebo.2. Immunohistochemical:The expression of protease activated receptor-2 on autologous full-thickness skin grafts with hyperpigmentation obviously higher than control groups.3. Western blot:the expression of protease activated receptor-2 on autologous full-thickness skin grafts with hyperpigmentation was obviously higher than control groups.4. The Realtime qPCR:the gene expression level of protease activated receptor-2 on autologous full-thickness skin grafts with hyperpigmentation were statistically significant difference vs control groups. (P< 0.05).5. Treated with the protease-activated receptor-2 activation inhibitor soybean trypsin inhibitor on skin grafts, the melanin mean IOD were obviously lower than control groups.Conclusion1. Using autologous full-thickness skin grafting on three different coloursguinea pig groups, observing the processing of hyperpigmentation to find out there were obviously hyperpigmentation on autologous full-thickness skin graft of the black guinea pigs after 16weeks. Black guinea pigs were ideal model about autologous full-thickness skin grafting with hyperpigmentation.2. General observation, masson-fontana stain, immunohistochemistry, western blotting and quantitative real-time polymerase chain reaction were carried out to detect the skin grafts colour change, the amount of melanin,the expression of protease-activated receptor-2 in autolougous full-thickness skin grafts after operation.We find out the amount of melanin and expression of protease-activated receptor-2 were much higher than control groups.Treated with protease-activated receptor-2 activation inhibitor soybean trypsin inhibitor four weeks. The amount of melanin in autologous full-thickness skin grafts of A group was lower than that in control treated with placebo.3. There is positive correlation between the expression level of protease-activated receptor-2 and autologous skin grafts with hyperpigmentation and that protease-activated receptor-2 overexpression of may play an important role in the hyperpigmented process of in autologous full-thickness skin grafts. This study may provides a novel mechanism for hyperpigmentation in. autologous full-thickness skin grafts.