体外培养的人恶性黑色素瘤球体细胞干细胞特性观察

摘要

Objective To observe the stem cell properties of human malignant melanoma cells cultured in serum-free me-dium.Methods Human malignant melanoma A375 cell line was cultured in serum-free medium, and the melanoma sphere cells (sphere group) and common adherent cells (adherent group) were obtained.Cell migration was detected using Transwell cham-ber.CD20 and CD133 were detected by immunofluorescence.Sixteen BALB/c mice were divided into the experimental group and control group (8 rats in each group), and the sphere cells and the adherent cells of 1 mL (104 ) were injected into the mouse shoulder blade by subcutaneous injection in the sphere and the adherent groups, respectively.The tumor cells were observed and compared between the two groups.Six weeks later, the mice were killed to obtain the tumor tissues.Results A375 cells were seeded in serum-free medium, visible small round ball suspension cells formed and gradually becomes larger , the ball inside the cells were closely connected with good refraction.The cell migration ability and the expression intensity of CD20 and CD133 in the sphere group were higher than those in the adherent group .After 6 weeks of injection, the mice in the experimental group had a large tumor, and no tumor growth was found in the control group.The tumor tissues of the experimental group were stained with HE, and the malignant melanoma was pathologically diagnosed.Conclusions Human malignant melanoma cells cultured in serum-free medium have the characteristics of stem cells with high migration ability and tumorigenesis .High expression of im-mune markers CD20, CD133 is found and malignant melanoma exists in the tumor stem cells.%目的：观察体外培养的人恶性黑色素瘤球体细胞的干细胞特性。方法利用无血清培养基培养人恶性黑色素瘤A375细胞系，获得黑色素瘤球体细胞（球体组）和普通贴壁细胞（贴壁组）。采用Transwell小室检测两组细胞迁移能力。采用免疫荧光法检测两组细胞中的CD20、CD133。取BALB／c小鼠16只，分实验组和对照组（各8只），于小鼠肩胛骨处皮下分别注射球体组、贴壁组细胞各1 mL（104个细胞），观察并比较两组细胞致瘤能力。6周后处死小鼠，取瘤。结果 A375细胞接种于无血清培养基后，可见小的类圆形悬浮细胞球形成并逐渐变大，球内细胞连接紧密，折光性好。球体组细胞迁移能力及细胞中CD20、CD133表达强度均高于贴壁组。注射细胞6周后，实验组小鼠均长出较大肿瘤，对照组小鼠均未见肿瘤生长。取实验组小鼠的肿瘤组织行HE染色，病理诊断为恶性黑色素瘤。结论体外培养的人恶性黑色素瘤球体细胞具有干细胞特性，迁移能力和致瘤能力强，高表达免疫标记CD20、CD133，恶性黑色素瘤存在肿瘤干细胞。