中华皮肤科杂志 ›› 2014, Vol. 47 ›› Issue (11): 767-771.

• 论著 •    下一篇

Notch-RBP-J信号通路对小鼠黑素瘤细胞B16F10与小鼠单核巨噬细胞RAW264.7共培养巨噬细胞极化影响的研究

吴琼1,陈浩2,周武庆1,1,李阿梅3,吕雅琳1,胡彬1,姜祎群2,孙建方2   

  1. 1. 中国医学科学院北京协和医学院皮肤病研究所
    2. 南京 中国医学科学院北京协和医学院皮肤病研究所
    3. 南京市 中国医学科学院皮肤病研究所
  • 收稿日期:2013-12-23 修回日期:2014-06-16 出版日期:2014-11-15 发布日期:2014-11-01
  • 通讯作者: 姜祎群 E-mail:yiqunjiang@qq.com
  • 基金资助:
    国家自然科学基金;中国医学科学院北京协和医学院青年基金

Effect of the Notch-RBP-J signaling pathway on the polarization of RAW264.7 murine macrophage-like cells cocultured with B16F10 murine melanoma cells

  • Received:2013-12-23 Revised:2014-06-16 Online:2014-11-15 Published:2014-11-01
  • Contact: Yi-Qun JIANG E-mail:yiqunjiang@qq.com

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摘要： 目的 探讨小鼠黑素瘤细胞B16F10与小鼠单核巨噬细胞RAW264.7共培养中Notch-RBP-J信号通路对巨噬细胞表型分化的影响。 方法 设计针对CBF1/RBP-Jκ基因的siRNA编码序列，转染至小鼠单核巨噬细胞RAW264.7细胞。实验分4组：沉默前共培养组，B16F10细胞与RAW264.7细胞共培养；沉默后共培养组，B16F10细胞与RBP-Jκ基因沉默后的RAW264.7细胞共培养；空白对照组，单独培养的RAW264.7细胞；阳性对照组，白细胞介素4刺激诱导的RAW264.7细胞。Western印迹法、ELISA法及流式细胞仪检测各组巨噬细胞的表型；RT-PCR法检测各组notch1、notch2、DLL1、DLL4及Hes1的表达。采用SPSS17.0软件进行重复测量方差分析及单因素方差分析、线性趋势检验、Bonferroni两两比较。 结果 Western印迹结果显示，RAW264.7与B16F10共培养不同时间后，RAW264.7细胞的CD163在空白对照组、共培养24 h、48 h、72 h组、阳性对照组的相对表达量分别为1.016 ± 0.018、1.274 ± 0.034、2.065 ± 0.094、3.615 ± 0.144、3.099 ± 0.071，经单因素方差分析（n = 4），F = 527.42，P < 0.01，共培养后RAW264.7细胞CD163表达量较空白对照组增加；ELISA检测结果显示，共培养24、48、72 h，RAW264.7细胞培养上清液白细胞介素10的水平分别为（167.610 ± 3.527）、（433.433 ± 5.558）、（679.673 ± 8.101） ng/L。沉默后共培养24、48、72 h，RAW264.7细胞培养上清液白细胞介素10表达量分别为（63.403 ± 0.856）、（103.427 ± 2.072）、（202.297 ± 3.610） ng/L，较未沉默时降低（F = 8.01，P < 0.05）。Western印迹法及流式细胞仪分别检测沉默后共培养RAW264.7细胞CD163、CD206的表达量，均较未沉默时减少（P < 0.05）。RT-PCR示，沉默后共培养组较沉默前共培养组及对照组的Notch信号通路相关基因mRNA表达均降低。 结论 沉默Notch-RBP-J信号通路后共培养组中RAW264.7细胞向M2型极化较未沉默共培养组减少，总体表型仍为M2型；该通路的活化促进RAW264.7细胞向M2型极化。

关键词: Notch-RBP-J信号通路, 巨噬细胞极化, 共同培养技术

Abstract: Wu Qiong, Chen Hao, Zhou Wuqing, Li Amei, Lyu Yalin, Hu Bin, Jiang Yiqun, Sun Jianfang. Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China Corresponding authors: Jiang Yiqun, Email: yiqunjiang@qq.com; Sun Jianfang, Email: sunjf57@163.com 【Abstract】 Objective To investigate the effect of the Notch-RBP-J signaling pathway on the phenotypic differentiation of RAW264.7 murine macrophage-like cells cocultured with B16F10 murine melanoma cells. Methods A small interference RNA （siRNA） targeting the CBF1/RBP-Jκ gene was designed. RAW264.7 murine macrophage-like cells were divided into four groups: unsilenced co-culture group cocultured with B16F10 cells, silenced co-culture group transfected with the designed siRNA and cocultured with B16F10 cells, blank control group cultured alone, positive control group induced by interleukin-4 （IL-4）. After additional culture for different durations, Western blot, enzyme-linked immunosorbent assay （ELISA） and flow cytometry were conducted to determine the phenotype of macrophages, and reverse transcription （RT）-PCR was performed to detect the expressions of notch1, notch2, DLL1, DLL4 and Hes1 genes in macrophages. Statistical analysis was carried out by one-way analysis of variance （ANOVA）, repeated measures ANOVA, linear trend test and the Bonferroni method with the SPSS17.0 software. Results Western blot showed that the relative expression level of CD163 in RAW264.7 cells was 1.016 ± 0.018 in the blank control group, 1.274 ± 0.034, 2.065 ± 0.094 and 3.615 ± 0.144 in the unsilenced co-culture group at 24, 48 and 72 hours respectively, and 3.099 ± 0.071 in the positive control group, with significant differences between these groups （n = 4, F = 527.42, P < 0.01）. There was a significant increase in CD163 expression in RAW264.7 cells in the unsilenced co-culture group compared with the blank control group. As ELISA revealed, the levels of IL-10 in the culture supernatant of RAW264.7 cells were （167.61 ± 3.527）, （433.433 ± 5.558） and （679.673 ± 8.101） ng/L in the unsilenced co-culture group at 24, 48, and 72 hours respectively, significantly higher than those in the silenced co-culture group （（63.403 ± 0.856）, （103.427 ± 2.072）, （202.297 ± 3.61） ng/L, respectively, F = 8.01, P < 0.05）. Western blot and flow cytometry both demonstrated a statistical reduction in the expressions of CD163 and CD206 in RAW264.7 cells in the silenced co-culture group compared with the unsilenced co-culture group （both P < 0.05）. The mRNA expressions of notch signaling pathway-related genes in RAW264.7 cells were also attenuated in the silenced co-culture group in comparison with the unsilenced co-culture group. Conclusions Although most of the RAW264.7 cells in the silenced co-culture group exhibited the M2 phenotype, their polarization toward M2 phenotype was weakened compared with those in the unsilenced co-culture group, implying that the activation of the Notch-RBP-J signaling pathway promotes the M2-polarization of RAW264.7 cells.

Key words: Notch-RBP-J signaling pathway, Macrophage polarization, Coculture techniques