Establishment of melanoblast cell lines and analysis of the factor involving melanocyte development

干细胞因子（SCF）和内皮素-3（ET 3）都是黑素细胞发育所必需的。为了获得可以在没有饲养细胞的情况下存活的成黑素细胞的永生细胞群，我们首先通过用补充有SCF和ET 3的培养基孵育从WB品系的S1/+和+/+小鼠获得神经嵴细胞的永生细胞群，并将它们命名为NCC-SE 3细胞。NCC-SE 3细胞呈双极、多角形或圆形，具有I-III期黑素小体（主要为I期）。免疫组化染色显示，它们对二羟苯丙氨酸（DOPA）反应阳性，表达酪氨酸激酶受体（KIT）、酪氨酸酶、酪氨酸酶相关蛋白1（TRP 1）、酪氨酸酶相关蛋白2（TRP 2）和内皮素B受体（ETR B）。接下来，我们通过将培养基从补充有SCF+ ET 3的培养基改变为补充有ET 3的SCT的培养基来培养NCC-SE 3细胞。单独用ET 3培养的NCC-SE 3细胞，称为NCC-E3细胞，形状为双极，主要具有II期黑素体，并表达与NCC-SE 3细胞相同的蛋白质。然而，与SCF单独培养的NCC-SE 3细胞，命名为NCC-S4.1细胞，在形状上是多边形的，并且主要具有I期黑素体。他们被认为更不成熟，因为他们对KIT、TRP 1和TRP 2呈阳性，但对ETRB、酪氨酸酶和DOPA反应不呈阳性。当加入12-o-十四酰-13-乙酸酯（TPA）和霍乱毒素后，NCC-S4.1细胞形态由多边形变为双极形，并呈DOPA阳性。这表明NCC-S4.1细胞是具有分化成黑素细胞的潜力的成黑素细胞。这些细胞群对于研究影响黑素细胞发育和黑素生成的因素将是非常有用的。

Stem cell factor (SCF) and endothelin-3 (ET3) are both necessary for melanocyte development. In order to obtain immortal cell populations of melanoblasts which can survive without feeder cells, we first obtained an immortal cell population of neural crest cells form S1/+ and +/+ mice of strain WB by incubating with a culture medium supplemented with SCF and ET3, and designated them as NCC-SE3 cells. NCC-SE3 cells were bipolar, polygonal or round in shape and possessed stage I-III melanosomes (mainly stage I). They were positive to dihydroxyphenylalanine (DOPA) reaction and expressed KIT (a receptor tyrosin kinase), tyrosinase, tyrosinase related potein-1 (TRP1), tyrosinase related potein-2 (TRP2), and endothelin B receptor (ETRB) as determined by immunostaining. We next cultured NCC-SE3 cells by changing culture medium from the one supplemented with SCF+ET3 to the one supplemented with SCT of ET3. NCC-SE3 cells cultured with ET3 alone, designated as NCC-E3 cells, were bipolar in shape and had mainly stage II melanosomes and expressed the same proteins as did NCC-SE3 cells. However, NCC-SE3 cells cultured with SCF alone, designated as NCC-S4.1 cells, were polygonal in shape and had mainly stage I melanosomes. They are thought to be more immature, because they were positive to KIT, TRP1, and TRP2 but not to ETRB, tyrosinase, and DOPA reaction. When 12-o-tetradecanoy1-13-acetate (TPA) and cholera toxin were added to the culture medium, NCC-S4.1 cells changed the shape from polygonal to bipolar and became DOPA positive. This suggests that NCC-S4.1 cells are melanoblasts that have the potential to differentiate into melanocytes. These cell populations will be extremely useful to study factors which affect melanocyte development and melanogenesis.