The Biological Characteristics Of Mice Melanocytes Isolated And Cultured In Vitro

The types, quantity and distribution of melanins, produced by melanocytes, are responsible for the hair and coat color. To get the ideal melanocytes for the research of hair color formation and pigmentation, the melanocytes lines was isolated and cultured from mice skin and the biological characteristics were analyzed by several methods in this study.Testing and the results were as follows:1. The primary melanocytes were isolated by 0.25% Dispase II enzyme and 0.25% trypsin/0.02% EDTA digestion, which showed typical characteristics with multi-dendritic or bipolar shape, thin and long dendritic cells and special outgrowth.2. Dopa staining were found in the cytoplasm, dendrite and nuclei of melanocytes from different passages (4,7,10 and 13) of mice melanocytes. And the positive immunocytohistochemical signals were found for S-100, MITF, TYR, TYRP1, TYRP2 in melanocytes. All the above supported the melanogenic traits of melanocytes.3. qRT-PCR results showed that MITF, TYR, TYRP1 mRNA expression in melanocytes from passages7 and 10 was a little different, compared to melanocytes from passage 4, but significantl difference appeared in melanocytes from passage 13 with high expression of TYR and TYRP1, which is the marker in melanoma.4. The cell adhesion and proliferation was detected by iCelligence real-time cell analysis systemand the results showed that the melanocytes from different passages began adherent proliferation after 2 h of culture, the proliferation of melanocytes from passage 4,7,10 of cells reached peak after 40 h of culture, and then decreasing. While the melanocytes from passage 13 showed the stronger proliferation until 100 h of culture.5. a-MSH was added in culture medium when melanocytes from passage 13 were cultured. Compared to the control, MITF、TYR and TYRP2 mRNA expression was 1.528 folds,4.623 folds and 1.489 folds when they are cultured for 72 h.Conclusion:the melanocytes from passage at least 10 were ideal material for research on hair color formation and pigmentation, because the melanocytes showed the melanoma characteristics with strong proliferation and TYR high expression.