Study of transforming growth factor β in melanocyte proliferation and differentiation of mouse neural crest cells via stem cell factor/KIT signaling

干细胞因子对黑素细胞在胚胎发育过程中的迁移和分化是必不可少的，这是因为观察到干细胞因子基因或其配体KIT的突变会导致小鼠毛皮色素沉着缺陷。转化生长因子β1参与了细胞增殖和分化的调控。NCC-Melb4是从小鼠NCC中克隆的永生细胞系。NCC-Melb4细胞为研究黑素细胞分化和增殖的特定阶段提供了模型。它们还表达KIT作为黑素母细胞标志物。以NCC-MELB4细胞株为研究对象，研究转化生长因子β-1对未成熟黑素细胞前体细胞分化和增殖的影响。免疫组织化学显示NCC-MALB4细胞表达转化生长因子β-1。抗转化生长因子β-1抗体抑制细胞生长，下调KIT蛋白和基因表达。为了进一步研究自分泌转化生长因子β1的激活作用，将NCC-Melb4细胞与外源性转化生长因子β1培养液孵育。KIT蛋白随着抗转化生长因子β-1抗体浓度的升高而降低，呈剂量依赖性。结论：转化生长因子β1可通过自分泌和/或旁分泌调节促进黑素细胞前体细胞的增殖。我们利用野生型小鼠原代培养系统进一步研究了转化生长因子β-1对体外培养的影响。抗转化生长因子β-1抗体使KIT阳性的NCC数量减少。此外，野生型神经脊组织块内供应的抗转化生长因子β-1抗体可抑制NCC的生长。这些结果表明，转化生长因子β1以自分泌/旁分泌的方式影响黑素细胞前体细胞的增殖和分化。

SCF is essential to the migration and differentiation of melanocytes during embryogenesis based on the observation that mutations in either the SCF gene, or its ligand, KIT, result in defects in coat pigmentation in mice. TGF β1 has been implicated in the regulation of both cellular proliferation and differentiation. NCC-melb4, an immortal cloned cell line, was cloned from a mouse NCC. NCC-melb4 cells provide model to study the specific stage of differentiation and proliferation of melanocytes. They also express KIT as a melanoblast marker. Using the NCC-melb4 cell line, we investigated the effect of TGF β1 on the differentiation and proliferation of immature melanocyte precursors. Immunohistochemically, NCC-melb4 cells showed TGF β1 expression. The anti-TGF β1 antibody inhibited the cell growth, and downregulated the KIT protein and mRNA expression. To further investigate the activation of autocrine TGF β1, NCC-melb4 cells were incubated on-exogenous TGF β1 culture medium. KIT protein decreased with anti-TGF β1 antibody concentration in a dose-dependent fashion. We concluded that in NCC-melb4 cells, TGF β1 promotes melanocyte precursor proliferation in autocrine and/or paracrine regulation. We further investigated the influence of TGF β1 in vitro using a NCC primary culture system from wild-type mice. Anti-TGF β1 antibody decreased the number of KIT positive NCC. I addition, the anti-TGF β1 antibody supplied within the wild-type neural crest explants abolished the growth of NCC. These results indicate that TGF β1 affect melanocyte precursor proliferation and differentiation in the presence of SCF/KIT in an autocrine/paracrine manner.