| Mammalian body surface is covered with skin and hair, in which the depth change and the difference of the hair color is affected by the external environment, nutritional intake, genetic inheritance and so on, yet how much melanin synthesis directly decided the hair color. The study of Pigmentation found that endothelin (Endothelin, EDN) as Paracrine synthesized by the keratinocytes play a key role in the immature melanocytes of differentiation and migration or promoting mature melanocytes growth. Even so, the impact of sheep hair melanin synthesis has not been reported and the effect of different pigment cells remains to be studied. In this study, different coat color of sheep and different melanoma cells were taken as the research object, using a variety of biological methods to explore the mechanism of EDN3 on the formation of pigment-producing and its regulatory role in different coat color sheep.(1) The relative expression of EDN3 in different sheep skin coat color by real-time PCR analysis showed, the expression of EDN3 in white sheep skin was 4.8 times of friesian sheep black skin, and was 5.5 times of all black sheep skin of expression. The expression of EDN3 in white friesian sheep skin was 3.8 times of Friesian sheep black skin, and was 4.3 times of black sheep skin. The EDN3 mRNA level expressed in the white sheep and black sheep skin was extremely significant.(2) ELISA assay was used to detect the EDN3 protein expression in different coat color of sheep skin, the results showed:all white sheep skin expression of EDN3 was 4.5 times that of black Friesian sheep skin, and was 5.1 times of all black sheep skin expression level. The EDN3 expression of white skin in Friesian sheep was 3.3 times of black Friesian sheep skin, and was 3.7 times of black sheep skin. The EDN3 protein level expressed in the white sheep and black sheep skin was extremely significant.(3) The localization of EDN3 by immunohischemistry showed that the EDN3 expressed normally in black and white sheep skin, and the EDN3 was located in the matrix, outer root sheath and dermal papilla of hair follicular.(4) Melanocytes from black and white coat color were cultured in vitro, and MTT assay was used to detect the different cell proliferation rate in different concentrations of EDN3 affected(10-10 mol/L,10-9 mol/L,10-8 mol/L,10-7 mol/L,10-6 mol/L). The results showed that the proliferation rate of melanocytes under the stimulus of EDN3 had the opposite effect, and EDN3 in 10-8mol/L concentration was most efficient, then when the concentration was too high (10-7 mol/L,10-6 mol/L) it inhibited the growth rate in partial. Comparison of the proliferation of melanocytes displayed that the melanocytes derived white skin was significantly higher than the melanocytes from black skin.(5) The Real-time PCR showed that the mRNA expression of EDNRB, NRas, TYR had changed when the concentration of EDN3 was 10-8 mol/L. The results showed that:the expression of the receptor EDNRB and downstream genes NRas mRNA of EDN3 has increased significantly, but the expression of TYR mRNA increased significantly only in black skin-derived melanoma cells, while had no difference in white skin melanocytes.(6) Western Blot was used to detect EDNRB, NRas, TYR protein expression, and the results showed: the receptor EDNRB and downstream NRas protein expression were significantly increased, but the protein expression of TYR only increased significantly in black skin melanin cells, while the melanocytes of white skin had no significantly change.The above results showed: EDN3 could normally expressed in different coat color of sheep skin, and there were significant differences in expression. EDN3 promoted white source melanoma cell proliferation but had no effect on the rate-limiting enzyme TYR; EDN3 promoted black source melanoma cell proliferation and might promote the formation of melanin. |
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