Le Poole I C, van den Berg F M, van den Wijngaard R M, Galloway D A, van Amstel P J, Buffing A A, Smits H L, Westerhof W, Das P K
Department of Dermatology, Academic Medical Center, Amsterdam University, The Netherlands.
In Vitro Cell Dev Biol Anim. 1997 Jan;33(1):42-9. doi: 10.1007/s11626-997-0021-6.
Availability of a standard human melanocyte cell line with unlimited growth potential and otherwise normal melanocytic properties will greatly facilitate research in melanocyte biology and in vitro studies on the etiology of pigmentary disorders and melanoma. Using a retroviral vector, E6 and E7 open reading frames of human papilloma virus type 16 (HPV 16) have been introduced into cultured normal human melanocytes. Cells selected by increased resistance to geneticin conveyed by the vector and expressing E6E7 mRNA have been cloned to ensure genetic homogeneity. Since their establishment as primary cells, cloned PIG1 cells have undergone more than twice the amount of population doublings of senescent parental cells. Moreover, in passage numbers when parental cells had become senescent, proliferation of clonal cells was retained at levels exceeding those of normal human melanocytes in third passage by 100%. Further characterization has revealed that the cells remain dependent on tetradecanoyl phorbol 13-acetate (TPA) for growth and do not proliferate in soft agar nor form tumors in nude mice. The antigenic profile of the cells was slightly altered as compared to parental cells, but was incomparable to that of M14 melanoma cells. Importantly, PIG1 cells contain more melanin pigment than parental cells.
拥有具有无限生长潜力且黑素细胞特性正常的标准人类黑素细胞系将极大地促进黑素细胞生物学研究以及色素沉着障碍和黑色素瘤病因的体外研究。利用逆转录病毒载体，已将人乳头瘤病毒16型（HPV 16）的E6和E7开放阅读框导入培养的正常人黑素细胞中。通过对载体携带的遗传霉素抗性增加而筛选出并表达E6E7 mRNA的细胞已被克隆以确保基因同质性。自作为原代细胞建立以来，克隆的PIG1细胞经历的群体倍增次数比衰老的亲代细胞多两倍以上。此外，在亲代细胞已衰老的传代次数中，克隆细胞的增殖水平比第三代正常人黑素细胞高出100%。进一步的特征分析表明，这些细胞的生长仍然依赖十四酰佛波醇13 - 乙酸酯（TPA），在软琼脂中不增殖，在裸鼠中也不形成肿瘤。与亲代细胞相比，这些细胞的抗原谱略有改变，但与M14黑色素瘤细胞的抗原谱不同。重要的是，PIG1细胞比亲代细胞含有更多的黑色素。
