ANALYSIS OF REGULATIVE FUNCTION OF MELANIN-RELATED GENE EXPRESSION BY BIOCHEMICALAND CELLULAR BIOLOGICAL METHODS

通过比较黑鼠(C57BL/10JHir)和黑鼠(C57BL/10JHir-A/A)小鼠表皮黑素细胞在无血清原代培养中的增殖和分化活性，探讨agti(A)基因在表皮黑素细胞发育中的作用。刺鼠黑素细胞与黑色黑素细胞的增殖活性及对分化刺激因子的反应性无明显差异。但在电子显微镜下，培养的刺鼠黑素细胞比培养的黑色黑素细胞具有更多的“III”期黑素小体。相反，与黑色黑素细胞相比，刺鼠黑素细胞具有少量的IV期黑素小体。在刺鼠黑素细胞中，黑素小体从“III”到“IV”的成熟被认为是被抑制的。为了解粉染稀释液(P)基因对粉染稀释液(C57BL/10Hir-p/p)小鼠表皮黑素细胞增殖和分化的作用机制，从原代培养开始，将酪氨酸底物L酪氨酸(L-Try)添加到无血清的元细胞培养液中，观察其对黑素细胞增殖分化的影响。在粉眼稀释型黑素细胞中，经L-酪氨酸处理后，各期(“I”、“II”、“III”、“IV”)的黑素小体数量均显著增加，而对照组的“I”、“II”、“III”期黑素小体数量有限。培养细胞和条件培养液中吡咯-2，3，5-三羧酸和真黑素降解产物(PTCA)的含量均增加了2倍。这些结果表明，粉眼稀释基因参与调节黑素的合成和黑素小体的形成，影响L在黑素小体中的保留。

The role of the agouti(A) gene in the development of epidermal melanocytes by comparing the activity of proliferation and differentiation of epidermal melanocytes from black (C57BL/10JHir) and agouti (C57BL/10JHir-A/A) mice in serum-free primary culture. There was no significant difference between agouti and black melanocytes in the proliferative activity as well as the reactivity to differentiation-stimulating factors. However, cultured agouti melanocytes possessed numerous stage "III" melanosomes than cultured black melanocytes under the electron microscopic observations. On the contrary, agouti melanocytes possessed a small number of stage "IV" melanosomes as compared to black melanocytes. The maturation of melanosomes form stage "III" to "IV" in agouti melanocytes is thought to be inhibited. To understand the mechanism of the action of the pink-dyed dilution (p) gene on the proliferation and differentiation of the epidermal melanocytes of pink-dyed dilution mice (C57BL/10Hir-p/p),L-tyrosine(L-try), the substrate of tyrosine, was supplemented to a serum-free metanocyte growth medium from the initiation of primary culture, and its effects were examined. In pink-eyed dilution melanocytes cultured with L-tyr, the numbers of melanosomes in all stage("I","II","III",and "IV")were dramatically increased, despite the limited numbers of stage "I","II",and "III" melanosomes in control culture. The content of pyrrole-2,3,5-tricarboxylic and (PTCA, degradation product of eumelanin)both in cultured cells and conditioned media was increased 2-fold. These results suggest that the pink-eyed dilution gene is involved in regulating the melanin synthesis as well as melanosome formation affecting the retention of L-try in melanosomes.