中华皮肤科杂志 ›› 2014, Vol. 47 ›› Issue (9): 619-623.

• 论著 • 上一篇    下一篇

肿瘤相关巨噬细胞对皮肤恶性黑素瘤细胞A375生物学行为的影响

殷芳1,吴飞1,陈佳2,3,章楚光4,宋宁静1   

  1. 1. 上海市皮肤病医院
    2. 同济大学附属上海市皮肤病医院
    3. 南京 中国医学科学院北京协和医学院皮肤病研究所
    4. 上海市皮肤病性病防治中心
  • 收稿日期:2013-09-10 修回日期:2014-04-21 出版日期:2014-09-15 发布日期:2014-09-01
  • 通讯作者: 宋宁静 E-mail:songnj-10@hotmail.com
  • 基金资助:
    上海市自然科学基金;上海市卫生局科研课题计划

Effects of tumor-associated macrophages on the biological behavior of A375 human malignant melanoma cells

  • Received:2013-09-10 Revised:2014-04-21 Online:2014-09-15 Published:2014-09-01

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摘要： 目的 探讨肿瘤相关巨噬细胞对皮肤恶性黑素瘤细胞增殖、侵袭和迁移的影响。 方法 取对数生长期人单核细胞株U937，采用佛波酯诱导48 h后，分为3组， Mφ组（继续用佛波酯诱导48 h）、M1组（用25 mg/L LPS诱导48 h）、M2组（用15 μg/L IL-4诱导48 h）。酶联免疫吸附试验检测各组巨噬细胞上清中IL-12p70和IL-10表达水平。建立巨噬细胞与恶性黑素瘤A375细胞体外共培养体系，分别设单独培养组、Mφ组（A375细胞和Mφ巨噬细胞共培养）、M1组（A375细胞和M1型巨噬细胞共培养）、M2组（A375细胞和M2型巨噬细胞共培养），分别运用噻唑蓝法、Transwell小室法观察不同激活途径的巨噬细胞对A375细胞增殖、侵袭、迁移等生物学行为的影响。 结果 增殖实验显示，共培养48、72 h后Mφ巨噬细胞、M2型巨噬细胞显著促进A375细胞的增殖，M1型巨噬细胞抑制A375细胞的增殖，各处理组间两两比较，差异有统计学意义（均P < 0.05），共培养24 h后各组间两两比较A375细胞增殖率差异无统计学意义（P > 0.05）。侵袭实验显示M2组和Mφ组穿过Transwell膜的A375细胞数量（147.00 ± 7.92、113.22 ± 8.15）较单独培养组（84.11 ± 6.07）明显增加（P < 0.05），M1组穿膜细胞（56.44 ± 7.55）明显减少（P < 0.05）。迁移实验显示M2组和Mφ组穿过Transwell膜的A375细胞数量（198.33 ± 8.22、156.00 ± 8.83）较单独培养组（123.89 ± 7.01）明显增加（均P < 0.05），M1组穿膜细胞（97.11 ± 6.75）明显减少（P < 0.05）。 结论 IL-4激活的M2型巨噬细胞对A375细胞的增殖、侵袭和迁移可能起促进作用；脂多糖激活的M1型巨噬细胞对A375细胞的增殖、侵袭和迁移可能起抑制作用。佛波酯诱导的巨噬细胞更倾向于M2型巨噬细胞表型。

关键词: 黑色素瘤, 细胞系，肿瘤, 巨噬细胞, 细胞增殖, 细胞运动

Abstract: Yin Fang*, Wu Fei, Chen Jia, Zhang Chuguang, Song Ningjing. *Shanghai Skin Disease Hospital, Anhui Medical University, Shanghai 200443, China Corresponding author: Song Ningjing, Email: songnj-10@163.com 【Abstract】 Objective To evaluate the effects of tumor-associated macrophages on the proliferation, invasion and migration of human cutaneous malignant melanoma cells. Methods Cultured U937 human monocytic cells at logarithmic phase were classified into three groups to be pretreated with phorbol ester for 48 hours followed by 48-hour activation by phorbol ester （Mφ polarization）, lipopolysaccharide （LPS） at 25 mg/L （M1 polarization）, and interleukin （IL）-4 at 15 μg/L （M2 polarization） respectively. Then, enzyme-linked immunosorbent assay （ELISA） was performed to determine the levels of IL-12p70 and IL-10 in the supernatant of these activated cells. A375 human malignant melanoma cells were divided into four groups to be cultured alone or with Mφ-, M1- and M2-polarized macrophages respectively. After additional culture for different durations （24, 48 and 72 hours）, methyl thiazolyl tetrazolium （MTT） assay was conducted to estimate the proliferative activity, and Transwell assay to evaluate the invasion and migration activity, of the A375 cells. Results The proliferation of A375 cells was accelerated by coculture with Mφ- and M2-polarized macrophages, but inhibited by that with M1-polarized macrophages, with significant differences among the four groups in the proliferative activity at 48 and 72 hours （all P < 0.05）, but not at 24 hours （P > 0.05）. Invasion assay showed that the number of A375 cells that migrated through Transwell chambers was significantly larger in M2 and Mφ groups （147.00 ± 7.92 and 113.22 ± 8.15 respectively）, but smaller in the M1 group （56.44 ± 7.55）, than in the control group （84.11 ± 6.07, all P < 0.05）. Similarly, migration assay revealed a significant increase in the number of A375 cells that migrated through Transwell chambers in the M2 and Mφ groups （198.33 ± 8.22 and 156.00 ± 8.83 respectively）, but a significant decrease in the M1 group （97.11 ± 6.75） as compared with the control group （123.89 ± 7.01, all P < 0.05）. Conclusions The proliferation, invasion and migration of A375 cells can be accelerated by IL-4-activated M2-polarized macrophages, but decelerated by LPS-activated M1-polarized macrophages. Phorbol ester tends to induce monocytic cells to differentiate into M2-polarized macrophages.

Key words: Melanoma, Cell line, tumor, Macrophages, Cell proliferation, Cell movement