鼻咽癌细胞条件培养基诱导THP-1源巨噬细胞向M2型极化

Nasopharyngeal carcinoma cell conditioned medium induces THP-1-derived macrophages to polarize to M2

目的:探讨鼻咽癌细胞系(5-8F、HONE1)来源的条件培养基(CM)对THP-1来源巨噬细胞极化方向的影响。方法:分别用50 nmol/L、100 nmol/L、200 nmol/L佛波酯(PMA)刺激人单核细胞白血病THP-1细胞诱导分化为M0型巨噬细胞(THP-1-M0组),24 h后,加入10 pg/mL脂多糖(LPS)和20 ng/mL干扰素(IFN)-γ处理细胞72 h,使M0向类M1型细胞方向极化(THP-1-M1组);加入20 ng/mL IL-4和20 ng/mL IL-13处理THP-1-M0使其向类M2型方向极化(THP-1-M2组)。流式细胞术检测巨噬细胞标记物CD14、CD68、CD86、CD163和CD206的表达。制备鼻咽癌5-8F及HONE-1细胞的条件培养基,培养THP-1-M0细胞。采用实时荧光定量聚合酶链式反应(RT-qPCR)法检测白细胞介素(IL)-6、IL-10、肿瘤坏死因子(TNF)-α、转化生长因子(TGF)-β1基因表达,分析肿瘤细胞刺激M0巨噬细胞的极化方向。结果:100 nmol/L为PMA的最佳刺激浓度。THP-1-M1组CD68、CD86、CD163表达及IL-6、TNF-α、IL-10、TGF-β1基因表达上调,THP-1-M2组CD14表达下调,CD68、CD86、CD163、CD206表达及IL-10、TGF-β1基因表达上调(均P<0.05)。加入鼻咽癌细胞条件培养基后,TNF-α、TGF-β1表达降低,IL-10表达升高(均P<0.05)。结论:5-8F细胞条件培养基诱导THP-1源M0型巨噬细胞偏向M2表型极化,HONE1细胞条件培养基培养的M0型巨噬细胞极化为M1/M2混合表型。

Objective:To investigate the effects of conditioned medium(CM)derived from nasopharyngeal carcinoma cell lines(5-8F and HONE1)on the polarization direction of THP-1-derived macrophages.Methods:Human monocytes leukemia THP-1 cells were stimulated by 50 nmol/L,100 nmol/L and 200 nmol/L phorbol myristate acetate(PMA)to differentiate into M0 type macrophages(THP-1-M0 group).After 24 h,the cells were treated with 10 pg/mL lipopolysaccharide(LPS)and 20 ng/mL interferon(IFN)-γfor 72 h.M0 was polarized toward M1-like cells(THP-1-M1 group).Twenty ng/mL IL-4 and 20 ng/mL IL-13 were added to treat THP-1-M0 to polarize it toward M2-like direction(THP-1-M2 group).The expressions of macrophage markers CD14,CD68,CD86,CD163 and CD206 were detected by flow cytometry.The conditioned medium of nasopharyngeal carcinoma 5-8F and HONE-1 cells was prepared and THP-1-M0 cells were cultured.The gene expressions of interleukin(IL)-6,IL-10,tumor necrosis factor(TNF)-αand transforming growth factor(TGF)-β1 were detected by real-time reverse transcription-quantitative PCR(RT-qPCR),and the polarization direction of M0 macrophages stimulated by tumor cells was analyzed.Results:One hundred nmol/L is the best stimulation concentration of PMA.The expressions of CD68,CD86,CD163 and IL-6,TNF-α,IL-10,TGF-β1 genes were up-regulated in THP-1-M1 group,CD14 expression was down-regulated in THP-1-M2 group,CD68,CD86,CD163,CD206,IL-10 and TGF-β1 gene expressions were up-regulated in THP-1-M2 group(P<0.05).After adding nasopharyngeal carcinoma cell CM,the expressions of TNF-αand TGF-β1 decreased,while the expression of IL-10 increased(P<0.05).Conclusion:M0 macrophages derived from THP-1 were polarized to M2 phenotype by 5-8F cell CM,while M0 macrophages cultured in HONE1 cell CM showed M1/M2 mixed phenotype.