| Objective1. By observing the comparisons of microskin's morphology and cytoactive under different mincing time, to determine the best mincing time of microskin grafting.2. Inject in the back epidermis of nude mice with cultured human epidermal melanocytes which are in a certain concentration, then observe whether there are pigment areas or not, if they appear, whether the areas expand over time or not, and what is the maximum expansion range, etc. All of these are to accumulate theoretical basis for clinical application.MethodsPartl. Comparisons of microskin's morphology and cytoactive under different mincing time.Selected 8 patients randomly. Then split-thickness skin which size 5 cm2 of lateral thigh was chosen from each patients, the scale for thickness of electric dermatome was 0.25mm. Cut the skin into five equal parts, each part was 1 cm2. Four parts of the skin were minced into microskin, the mincing time was 5min, 10min,15min,20min, respectively. The remaining one was as a control group. Then, the determination of succinate dehydrogenase (SDH) and oxygen consumption in each group was carried out. After the determination, part of the tissue was sent for HE staining.Part2. Preliminary observation about skin histology of nude nice which were injected with human epidermal melanocytes (HEM).Cell culture experiment:purchased human epidermal melanocytes-dark (HEM-d) primary cells from the ScienCell company in United States, then resuscitated the cells and cultured, subcultured, cryopreserved, etc. Observed the morphology of primary and each generations of HEM-d by using inverted phase contrast microscope, meanwhile took photographs of them. The first batch of HEM-d were mainly used to observe and accumulate the experience of culture. The second batch of HEM-d, after the recovery of primary cells and subculturing to passage one, part of the cells were used to do animal experiments, the other part of the cells to continue subculture.Animal experiment:5 SPF grade male nude mice (BALB/c-nu) which were injected in the back epidermis with cultured HEM-d-P1, these cells were in the medium and its concentration was 4×104/ml; Set up a controlled trial that the epidermis was injected with complete medium of HEM-d. After the injection, continuously observed the skin pigmentation of nude mice whether it changed or not, and whether the pigment areas expanded over time or not; Respectively took the full-thickness skin that was injected with HEM-d-P1 at Id,15d,30d,60d. Put full-thickness skins into 4% paraformaldehyde fixative to be stored. Then made them stained by HE staining to observe whether there were melanocytes, melanin granules or not and their distribution under light microscope.Results1. There was an optimal time point for mincing skin into microskin, and the cytoactive of microskin would gradually decrease if the time was too long.2. It could make the nude mice skin black with intradermal injection in nude mice by using cultured human epidermal melanocytes. HE staining showed the presence of melanocytes which were in the skin of nude mice. Moreover the cells were alive, and the black pigment areas gradually expanded during the experimental period.Conclusions1. There was a most appropriate time for mincing skin into microskin. The particle size changed little with too long time of mincing, however the cytoactive of microskin would gradually decrease.2. It could make the nude mice skin black with intradermal injection in nude mice by using cultured human epidermal melanocytes, and the black pigment areas gradually expanded during the experimental period. HE staining showed that the morphology of melanocytes which in the injection area were normal. This provided a theoretical basis for clinical practice. |
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