关键词: 黑色素瘤, 细胞系, 肿瘤, 中国汉族人群, 淋巴结转移

Abstract: 【Abstract】 Objective To establish a melanoma cell line derived from a Chinese female patient of Han nationality with metastatic melanoma, and to study its basic biological characteristics. Methods Metastatic melanoma cells were isolated from axillary lymph nodes of a 17-year-old female patient with malignant melanoma, and subjected to culture in vitro to establish a cell line. Short tandem repeat （STR） genotyping was performed to compare the information about the cell line and its derived tissue, and to detect gene mutations; cell counting kit-8 （CCK8） assay was conducted to assess the cellular proliferative activity, and soft agar cloning assay to assess the anchorage-independent proliferation; karyotype analysis was performed to determine the number and structure of chromosomes; with the highly aggressive melanoma cell line A2058 and keratinocyte line HaCaT serving as controls, Transwell assay was conducted to detect differences in cell migratory and invasive abilities, cell immunofluorescence assay and Western blot analysis were performed to determine the protein expression of melanoma-specific markers HMB45, S100 and Melan-A; the tumorigenicity was evaluated in vivo in a tumor-bearing mouse model. Results A melanoma cell line was successfully established and named ZJMM-45, which was cultured for more than 70 passages over a 1-year period, and showed a stable shape and proliferative activity. The cells were spindle-shaped or polygonal, and could produce melanin. STR matching analysis showed that the ZJMM-45 sample was 96% matched with the cryopreserved lymph node tissue of the patient, suggesting that they were from the same source. A tumor-related gene BRAFV600E mutation （c.1799T>A） was identified in the ZJMM-45 cell line; karyotype analysis revealed that ZJMM-45 cells had triploid chromosomes and abnormal structures. ZJMM-45 cells grew exponentially in vitro and reached a plateau-phase in growth on day 5; the cells grew clonally and formed colonies in agar, showing anchorage-independent and malignant proliferative activity. Cell immunofluorescence assay and Western blot analysis showed that both ZJMM-45 and A2058 cells expressed HMB45, S100 and Melan-A; Transwell assay revealed that the numbers of invasive and migratory ZJMM-45 cells （300 ± 14, 260 ± 14, respectively） were significantly higher than those of invasive and migratory A2058 cells （150 ± 6, 160 ± 19, t = 13.25, 11.76, respectively, both P < 0.001）. The tumor-bearing mouse experiment showed that all 5 nude mice developed tumors with an inner diameter of 1.0 cm after 4 weeks, and the tumors were histopathologically characterized by proliferating hyperchromatic melanoma cell nuclei and formation of small nests, which were similar to primary solid tumors. Conclusion The metastatic melanoma cell line ZJMM-45 derived from a Chinese patient with melanoma was successfully constructed, which carried the BRAFV600E mutation and expressed melanoma-specific markers, and was characterized by rapid proliferation, invasion and metastasis in both in vivo and in vitro culture, as well as obvious tumorigenicity in the in vivo experiment.

Key words: Melanoma, Cell line, tumor, Chinese Han population, Lymphoid node metastasis