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目的:体外培养小胶质N9细胞并观察其条件培养液(conditioned medium,CM)对乳腺癌MCF-7细胞生存率的影响,以探讨小胶质细胞在脑转移癌中的作用机制。方法:混合培养N9细胞和MCF-7细胞,在倒置光学显微镜下观察细胞形态学的变化。采用脂多糖(lipopolysaccharide,LPS)、MCF-7细胞及其条件培养液MCF-7-CM刺激N9细胞获得N9-CM;ELISA法检测N9-CM中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-17(interleukin-17,IL-17)的表达,Griess体系检测一氧化氮(nitric oxide,NO)的生成量。将N9-CM按不同浓度(10%、25%、50%、75%和100%)作用于MCF-7细胞,MTT法检测N9-CM对MCF-7细胞生存率的影响。结果:MCF-7细胞与N9细胞共培养,N9细胞由静止态变为激活态;LPS、MCF-7细胞及其上清液均可以诱导N9细胞产生TNF-α、IL-17和NO;N9-CM浓度达50%～100%时对MCF-7细胞生长有抑制作用,且作用强度呈时间-浓度相关性。结论:小胶质细胞可以被转移到CNS的乳腺癌细胞激活并发挥肿瘤抑制作用。其作用强度与小胶质细胞分泌NO及细胞因子TNF-α和IL-17的浓度和作用时间呈正相关性。
OBJECTIVE: To investigate the effect of conditioned medium (CM) on the survival rate of breast cancer MCF-7 cells in vitro and to explore the mechanism of microglia in brain metastases. Methods: N9 cells and MCF-7 cells were mixed and cultured. The morphological changes of cells were observed under inverted light microscope. N9-CM was obtained by stimulating N9 cells with lipopolysaccharide (LPS), MCF-7 cells and its conditioned medium MCF-7-CM. The levels of tumor necrosis factor-α (TNF-α) and interleukin-17 (IL-17) were detected by enzyme linked immunosorbent assay (ELISA). The formation of nitric oxide (NO) was detected by Griess system. The effect of N9-CM on MCF-7 cell viability was tested by MTT assay at different concentrations (10%, 25%, 50%, 75% and 100%). Results: MCF-7 cells were co-cultured with N9 cells, and N9 cells changed from quiescent state to activated state. LPS, MCF-7 cells and their supernatants all induced N9 cells to produce TNF-α, IL-17 and NO The concentration of -CM up to 50% ~ 100% inhibited the growth of MCF-7 cells, and the effect intensity showed a time-concentration correlation. CONCLUSIONS: Microglial cells can be activated by breast cancer cells that have metastasized to the CNS and exert tumor suppression. The intensity of action was positively correlated with the concentration and duration of NO and cytokines TNF-α and IL-17 secreted by microglial cells.