胶质瘤-巨噬融合细胞的巨噬细胞极化特征及其对胶质瘤细胞增殖和侵袭能力影响的实验研究
Experimental study on the macrophage polarization characteristics of glioma-macrophage fusion cells and their impact on the proliferation and invasion capabilities of glioma cells
胡跃东 1王佩文 1张梦梦 1周贤喆 1刘福生 2米蕊芳1
作者信息
- 1. 首都医科大学,北京市神经外科研究所,北京 100070
- 2. 首都医科大学,北京市神经外科研究所,北京 100070;首都医科大学附属北京天坛医院神经外科学中心,北京 100070
- 折叠
摘要
目的 通过建立胶质瘤细胞与巨噬细胞的融合细胞模型,探究胶质瘤-巨噬融合细胞的巨噬细胞极化特征及其对胶质瘤细胞增殖、侵袭能力的影响.方法 应用小鼠来源的胶质瘤细胞GL261和巨噬细胞RAW264.7构建并筛选GL261xRAW264.7融合细胞.分别培养GI261、RAW264.7、GL261xRAW264.7融合细胞,收集细胞上清获得相应的条件培养基(CM,即GL261-CM、RAW264.7-CM、GL261xRAW264.7-CM).采用流式细胞术检测GL261xRAW264.7融合细胞的染色体含量及其与RAW264.7细胞的巨噬细胞标志物CD86、CD206的表达量;通过CCK-8、Transwell或划痕实验评估GL261xRAW264.7融合细胞与亲代细胞(即GL261、RAW264.7细胞)增殖、迁移能力的差异,以及GL261xRAW264.7-CM与亲代细胞CM培养的GL261细胞增殖、迁移、侵袭能力的差异;通过免疫磁珠-流式细胞术检测RAW264.7与RAW264.7xGL261融合细胞分泌巨噬细胞M1型细胞因子肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、IL-6和IL-23及M2型细胞因子IL-10、转化生长因子β1(TGF-β)的差异.结果 成功构建了 GL261xRAW264.7融合细胞,且经流式细胞术证实GL261xRAW264.7融合细胞的染色体含量明显增加(G1和G2/M峰均较亲代细胞右移).CCK-8增殖实验结果显示,GL261xRAW264.7融合细胞的增殖能力较GL261细胞强,但较RAW264.7细胞弱,差异均有统计学意义(均P＜0.05);Transwell实验显示,GL261xRAW264.7融合细胞的迁移能力均较亲代细胞增强,差异均有统计学意义(均P＜0.001).流式细胞术检测结果显示,GL261xRAW264.7融合细胞与RAW264.7细胞CD206表达率的差异具有统计学意义[(75.80±2.31)％对比(4.72±0.47)％,P＜0.001];而CD86表达率的差异无统计学意义[(4.27±0.40)％对比(4.18±0.41)％,P＞0.05].免疫磁珠-流式细胞术检测结果显示,GL261xRAW264.7融合细胞与RAW264.7相比,IL-10、TGF-β表达增多,TNF-α、IL-1β减少,差异均有统计学意义(均P＜0.01);而IL-6、IL-23的差异均无统计学意义(均P＞0.05).CCK-8、Transwell及划痕实验均显示,GL261xRAW264.7-CM较亲代细胞CM培养的GL261细胞增殖、迁移和侵袭能力均增强,差异均有统计学意义(均P＜0.01).结论 体外构建的GL261xRAW264.7融合细胞具有M2型巨噬细胞的免疫特征,且可促进胶质瘤细胞的增殖、迁移及侵袭能力.
Abstract
Objective To establish a fusion cell model between glioma cells and macrophages to explore the polarization characteristics of glioma-macrophage fusion cells and their effects on the proliferation and invasive capabilities of glioma cells.Methods Mouse-derived glioma cells GL261 and macrophages RAW264.7 were used to construct and screen the GL261xRAW264.7 fusion cells.GL261,RAW264.7,and GL261xRAW264.7 fusion cells were cultured separately to collect their supernatants,and the respective conditioned media were obtained(CM,namely GL261-CM,RAW264.7-CM,GL261xRAW264.7-CM).Flow cytometry was employed to detect the chromosomal content of GL261xRAW264.7 fusion cells and their expression levels of macrophage markers CD86 and CD206.The differences in proliferation and migration abilities between GL261xRAW264.7 fusion cells and their parent cells(GL261 and RAW264.7 cells)were evaluated using CCK-8,Transwell,and scratch assays.Additionally,the effects of GL261xRAW264.7-CM,compared with parent cell CMs,on the proliferation,migration,and invasion capabilities of cultured GL261 cells were assessed.Immunomagnetic bead flow cytometry was utilized to detect differences in the secretion of macrophage M1-type cytokines TNF-α,IL-1β,IL-6,and IL-23 and M2-type cytokines IL-10,TGF-βbetween RAW264.7 and GL261xRAW264.7 fusion cells.Results The GL261xRAW264.7 fusion cells were successfully constructed,and flow cytometry confirmed a significant increase in their chromosomal content(right shifts in G1 and G2/M peaks compared to parent cells).CCK-8 assays showed that the proliferation ability of GL261xRAW264.7 fusion cells was stronger than that of GL261 and weaker than that of RAW264.7 with significant differences(all P＜0.05).Transwell assays demonstrated that the migration ability of GL261xRAW264.7 fusion cells was enhanced compared with the parent cells with significant differences(all P＜0.001).Flow cytometry revealed statistically significant differences in the expression rate of CD206 between GL261xRAW264.7 fusion cells and RAW264.7 cells[(75.80±2.31)％vs.(4.72±0.47)％,P＜0.001].However,there were no significant differences in CD86 expression[(4.27±0.40)％vs.(4.18± 0.41)％,P＞0.05].Immunomagnetic bead flow cytometry showed that compared with RAW264.7,GL261xRAW264.7 fusion cells exhibited increased expression of IL-10 and TGF-β,decreased expression of TNF-α and IL-1β with significant differences(all P＜0.01).However,there were no significant differences in the expression of IL-6 and IL-23(all P＞0.05).CCK-8,Transwell and scratch assays all indicated that the proliferation,migration and invasion of GL261 cells cultured in GL261 xRAW264.7-CM were enhanced compared with those cultured in parent cell CM with significant differences(all P＜0.01).Conclusion The in vitro constructed GL261xRAW264.7 fusion cells possess M2-type macrophage immune characteristics and can promote the proliferation,migration and invasion capabilities of glioma cells.
关键词
神经胶质瘤/细胞融合/细胞增殖/肿瘤相关巨噬细胞/细胞侵袭Key words
Glioma/Cell fusion/Cell proliferation/Tumor-associated macrophages/Cell invasion引用本文复制引用
基金项目
北京市自然科学基金(7222019)
北京市自然科学基金(7182026)
出版年
2024
NETL
NSTL
万方数据