Hirobe T
Division of Biology and Oncology, National Institute of Radiological Sciences, Anagawa, Chiba, Japan.
J Investig Dermatol Symp Proc. 2001 Nov;6(1):25-31. doi: 10.1046/j.0022-202x.2001.00001.x.
Mouse epidermal melanoblasts preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in serum-free melanoblast-defined medium (MDM). After 14 d, almost all keratinocytes that existed predominantly in the early stage of primary culture died, and pure cultures of melanoblasts were obtained. Epidermal melanoblasts dramatically increased in number in MDMDF consisting of MDM supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and basic fibroblast growth factor (bFGF). Epidermal melanocytes increased in number in MDMD consisting of MDM supplemented with DBcAMP. On the other hand, epidermal melanocytes were induced to differentiate in MDMM consisting of MDM supplemented with alpha-melanocyte-stimulating hormone (MSH). Pure cultured primary melanoblasts or melanocytes in MDMDF or MDMD were further cultured with MDMDF or MDMD supplemented with endothelin (ET)-1, -2, or -3 from 14 d. A dramatic increase in the number of melanoblasts or melanocytes was observed after 7 d; however, no increase in the number of melanoblasts or melanocytes was observed in the absence of ET-1, -2, or -3. The increase in the number of melanoblasts or melanocytes was comparable with that of melanoblasts or melanocytes cocultured with secondary keratinocytes in MDMDF or MDMD. Also, pure cultured primary melanoblasts in MDM were further cultured with MDMM supplemented with ET-1, -2, or -3 from 14 d. A dramatic increase in the percentage of melanocytes in the melanoblast-melanocyte population was observed after 7 d; however, no increase in the percentage of melanocytes was observed in the absence of ET-1, -2, or -3. The increase was comparable with that of melanocytes cocultured with secondary keratinocytes in MDMM. Moreover, anti-ET-1, -2, and -3 antibodies inhibited both the proliferation of melanoblasts or melanocytes in MDMDF or MDMD and the differentiation of melanocytes in MDMM in primary culture. These results suggest that ET-1, -2, and -3 are one member of the keratinocyte-derived factors that are involved in regulating the proliferation and differentiation of mouse epidermal melanocytes in primary culture.
在无血清黑素母细胞限定培养基（MDM）中，源自新生小鼠皮肤的分散表皮细胞悬液中的小鼠表皮黑素母细胞优先增殖。14天后，原代培养早期主要存在的几乎所有角质形成细胞死亡，从而获得了黑素母细胞的纯培养物。在补充有二丁酰腺苷3':5'-环磷酸（DBcAMP）和碱性成纤维细胞生长因子（bFGF）的MDM组成的MDMDF中，表皮黑素母细胞数量急剧增加。在补充有DBcAMP的MDM组成的MDMD中，表皮黑素细胞数量增加。另一方面，在补充有α-黑素细胞刺激素（MSH）的MDM组成的MDMM中，表皮黑素细胞被诱导分化。MDMDF或MDMD中的纯培养原代黑素母细胞或黑素细胞在14天后用补充有内皮素（ET）-1、-2或-3的MDMDF或MDMD进一步培养。7天后观察到黑素母细胞或黑素细胞数量急剧增加；然而，在没有ET-1、-2或-3的情况下未观察到黑素母细胞或黑素细胞数量增加。黑素母细胞或黑素细胞数量的增加与在MDMDF或MDMD中与二代角质形成细胞共培养的黑素母细胞或黑素细胞的增加相当。此外，MDM中的纯培养原代黑素母细胞在14天后用补充有ET-1、-2或-3的MDMM进一步培养。7天后观察到黑素母细胞-黑素细胞群体中黑素细胞百分比急剧增加；然而，在没有ET-1、-2或-3的情况下未观察到黑素细胞百分比增加。这种增加与在MDMM中与二代角质形成细胞共培养的黑素细胞的增加相当。此外，抗ET-1、-2和-3抗体在原代培养中抑制了MDMDF或MDMD中黑素母细胞或黑素细胞的增殖以及MDMM中黑素细胞的分化。这些结果表明，ET-1、-2和-3是角质形成细胞衍生因子的一员，参与调节原代培养中小鼠表皮黑素细胞的增殖和分化。
