The Effects Of Endothelin-1(EDN1) On Melanin Synthesis Of Sheep Skin Melanocytes In Vitro

The colour difference of mammals mainly depends on the number of melanocytes, melanin content and the distribution of melanosomes producted and transferred from melanocytes around the keratinocytes. On mammals, many researches on colour showed that many genes could influence the growth and migration of melanocytes. Therefore, melanocytes will lay the foundation for the research of the colour gene. EDN1 is one of the members of the endothelin family, and it has the effect on promoting mitosis and synthesis of melanin for melanocytes. In melanocytes, Combined with G-protein coupled receptor, EDN1 activates a series of signaling pathways. The culture in vitro and identification methods of sheep skin melanocytes were explored to lay a good foundation for establishing sheep skin melanocyte lines, and provide an ideal biological material for studying of genetic function of the colour. The research adopted the two-step digestion method (Dispase â…¡ enzyme and trypsin/EDTA) to obtain the sheep skin melanocytes. Then melanocytes were cultured, and their biological characteristics were identificated. Melanocytes were cultured with Me1M containing different concentrations of END1(10-10 mol/L,10-9 mol/L,10-8 mol/L and 10-7 mol/L), then the effect of EDN1 for proliferation of melanocytes, melanin synthesis, and the expression of relative genes were detected. Further, the effect of EDN1 for melanin synthesis in sheep skin melanocyte was anasysised. The main research contents and results were as follows.1. After primitive culture and subculture, morphology of melanocytes at various stages were observed. The results showed that within plating 12 h, melanocytes were adhered to the culture plate between keratinocytes (Figure 1A). After plating 24 h, most of melanocytes were multiple (Figure 1B). After 7 d, cell alignment was about 80%, and there were the typical dendritic structure.2. The cell growth curve was drawn by MTT method to study the proliferation ability. When cultured for 1-2 days, cells were growing slowly. At day 3, cells turned to the logarithmic phase. Subsequently, the confluence of cells became approximately 80%. But cells became slow and were in lag phase at day 7 (Figure 2).3. The cultured cells biology was identified by Dopa-staining, immunocytochemical localization of melanocyte markers and RT-PCR. Cells showed normal morphology and strong proliferation ability. Dopa-staining, immunocytochemistry and RT-PCR revealed that the function of cell biology were normal.4. After 72 h for adding EDN1, melanocytes were two or three dendritic in the control group, butdendrites were not obvious (Figure A), but the dendrites were obvious in the experimental group (Figure B,C,D).5. The proliferation rate of melanocytes were examined by MTT. The results showed that, compared tothe control group, the number of melanocytes was increased remarkably (P<0.05), and proliferation was the most obvious in 10"10 mol/L (P<0.01). 6. Compared to the control group, the melanin contents in melanocytes were increased remarkably(P<0.05), and proliferation was the most obvious in 10-10 mol/L (P<0.01). 7. The expression of genes (MITF, MC1R, TYR and EDNRB) was detected using qRT-PCR. Theresults analysised by SPSS 17.0 statistical analysis software showed that, compared with the control group,the expression of MITF, MC1R, TYR and EDNRB mRNA was 1.95,5.50,1.34 and 3.84 fold changes (P<0.05)In this study, the cultured system of melanocytes was established successfully. By studying the effectof EDN1 on proliferation of melanocytes, melanin synthesis and expression of related genes in vitro, wedrew a conclusion that EDN1 could regulate signal regulating pathways in sheep skin melanocytes andinfluence the formation of coat color.