The Dynamic Changes Of MC1R Positive Cells Expressing In The Process Of Skin Wound Healing

BackgroundSkin is the body’s important major organs and exposed to the surface, so it is the mostvulnerable to damage. According to incomplete statistics, China has500-1000patientsannually, who were just burnt. In those wounds,only a few are easy-to-heal, most ofthem are hard to heal or formed severe scar, seriously affecting the quality of life.Currently, most of the methods for wound healing are about promoting the rate ofhealing, the research about the regenerative medicine, which can promote tissueregeneration to achieve functional repair, is ignored. The stem cells in skin are the keyto regeneration of the tissue, and stem cell mobilization, proliferation, migration,differentiation mechanism is not clear to us.According to our preliminary studies andexperiments confirmed melanocyte receptors are highly expressed in stem cell-richregion,This is simularities that MSH/MC1R temporal and spatial expression pattern inthe wound healing process.So whether MC1R is expressed in epidermal stem cellsand whether it secretes MSH, thereby regulating epidermal stem cell proliferation,migration and differentiation, thus contributing to the healing of skin woundsbecomes an essential issue. The study is beginning with exploring thishypothesis.And the hypothesis has important clinical value and far-reaching socialsignificance in wound repairing, improving the quality of trauma patientsrehabilitation, reducing their disability rate.MethodThe study is divided into internal and vitro experiments and carried out fromthe different point of gene level,protein level,qualitative,positioning and quantitative.1.In vitro experiments:（1）Isolation and culture of neonatal rat skin epidermalstem cells,The epidermal stem cells dry marker K19, β1Integrin were stained withMC1R.Situ expression of epidermal stem cells are identified MC1R in the rat skin. （2）The cultured rat epidermal stem cell lysis total RNAwas extracted. WhetherMC1R is expressed in epidermal stem cells and whether it secretes MSH at thegenetic level by RT-PCR and sequencing PCR products.2. In vivo experiments: Choose6-8weeks old, healthy and clean SD male rats,then make wound model in the dorsal skin of the rats, thiedly, cut round wound andsurrounding2mm normal skin in the first1,3,5,7,14,21days after the death of the rats,and cut the skin in half. Freeze the half of the skin, which is obtained by the meansabove, in liquid nitrogen immediately, and crush it to extract the total of RNA in theskin. The detect the dynamic changes quantitatively of MC1R, stem cells gene:K19、β1Integrin in the process of the rat wound healing by the means of the quantitativePCR technique at the genetic level. The half of skin tissue wounds afterparaformaldehyde fixed paraffin-embedded sections were HE staining andimmunohistochemical staining.Expression was detected by in rat wound healingprocess MC1R positive cells expressing dynamic changes.Whether the expression ofMC1R detected in the skin stem cells by Immunofluorescence.Results1. The results of the cell Immunofluorescence shows: dry gene K19in ratepidermal stem cells, β1Integrin and MC1R all send a positive signal in the sameparts. Epidermal stem cells release MC1R, POMC, PC1, PC2,7B2in gene levels,which is detected by RT-PCR.And the amplified prodcuts were sequenced,In Pubmedin comparison with the original gene sequence overlap rate of99%.2. The wounds HE staining in the rat skin shows: the skin wound healingexperienced a period of bleeding phase, inflammation phase, cell proliferation phaseand tissue remodeling phase. Correspondingly, by MC1R immunohistochemicalstaining, we know that the positions, where MC1R appears, are different in differenttime during the time of wound healing. On the first day post-traumatic, the wound isin the inflammation phase. During this time, the inflammatory cells all move andgather to the wound edge, MC1R appears in the basal cells of the wound edges,inflammatory cells and the hair follicle in surrounding normal skin, while the positivesignal is not detected in the basal layer of the normal skin. On the first three days of the wound healing, the wound is in the cell proliferation phase. The tendency of thatbasal cells in the skin all move and gather to the wound edge can be observed clearlyin the HE staining. And in the immunohistochemical stainin MC1R in those cellsmigrating and gathering at the wound edge sends strong positive signal, and as whileas the wound heal, these cells are constantly extending and forming new basal layer ofskin.3.Wether the expression of MC1R detect epidermal stem cells in rat skin byimmunofluorescence.The results are that in epidermal stem cells, Dry K19sendsgreen signal, MC1R sends red signal, and the two can overlap in the same parts of thecell.4. From the result of the quantitative PCR, we know that during the process ofrat wound healing, MC1R, dry gene K19, β1Integrin all change as parabola. Fromthe first three days of the wound healing, the expression begins increasing gradually,while the expression gradually reduced to the normal levels, when the wounds of skinalmost heal.Conclusion1. Isolation and culture of rat skin tissue in vivo and in vitro epidermal stem cellsexpress MC1R。Therefore, further experiments are necessary, we need to stain morestem cells markers with MC1R, and verify our conjecture by different test methodfrom different points.2. During the process of the skin wound healing, the changes of the positionwhere MC1R ever appeared show that not only MC1R involved in the process, butalso the trend of the change is the same with the trend of stem cells change whileinvolving in wound healing. However, the question of whether the cells, which showpositive MC1R, are stem cells, and how MC1R migrate is not clear to us, so thefurther experiments are needed. What is more, the influences of MSH on woundhealing require the further internal and vitro experiments to explain.