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The aim of the present study was to establish an immunofluorescence method of antibody detection to identify melanocytes in the serum of vitiligo patients. Furthermore, we aimed to establish a method for the culture and proliferation of autologous pure melanocytes and to observe the effect of their transplantation for the treatment of vitiligo. Suspension of epidermal cells with melanocytes was performed using trypsin digestion of normal epiderm from eyelid operation and melanocytes were selectively cultured and proliferated in serum-free M2 medium. FITC-labeled rabbit anti-human antibody was used to detect the relative fluorescence intensity of the melanocytes. After identification with immunological and biological examinations, the melanocytes were transplanted to depigmented areas of vitiligo. Repigmentation was observed continuously. The results indicated that melanocytes could be selectively proliferated in the medium. Subsequently, pure melanocytes without contamination of fibroblast and keratinocyte were harvested. A total of 34 patients suffering vitiligo for between 3 months and 20 years with depigmented area (between 4 cm 2 and 70% of body surface) were divided into 19 cases of developing stage and 15 cases of stable stage, according to the change of depigmentation. A total of 15 developing cases were positive for the antibody against melanocytes, with the positive rate of 79%. The titers of serum was &gt;1:50 in 10 patients at the developing stage, and 5 developing patients were 1:10. Among the 15 stable cases, four were positive, with a positive rate of 27%. Fluorescence of antibody was localized in the cytoplasm of the melanocytes. Autologous melanocytes of vitiligo patients could be selectively proliferated in the medium. Next, pure melanocytes without contamination with fibroblasts and keratinocytes were harvested. A total of 16 vitiligo patients with 28 depigmented areas (2-200 cm 2 ) were treated with transplantation of melanocytes. Repigmentation of the transplanted areas appeared as red coloration after one month. All the vitiligous areas received transplantation were repigmented significantly with hypo- or hyper-pigmentation after 3-5 months. After 6-8 months, 87.5% of lesions showed repigmentation of &gt;50% of the lesion area. No scarring or other side-effects occurred. After follow-up of 5 years, no relapse was observed in transplantation area. Thus, an immunofluorescence method for the test of antibody to melanocytes in the serum of vitiligo patients was established. Transplantation of cultured autologous melanocytes was an effective and safe measure for treatment of vitiligo, particularly for patients with a large depigmented area.

Detection of auto antibodies and transplantation of cultured autologous melanocytes for the treatment of vitiligo