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目的建立肺癌细胞与单核/巨噬细胞间接共培养模型,观察在肺癌细胞条件培养基作用下抑制前体破骨细胞(RAW264.7)的CCR1对破骨细胞生成的影响。方法收集肺癌细胞A549的条件培养基,将条件培养基以不同浓度加入RAW264.7培养基中共培养,在共培养体系中加或不加CCR1特异性拮抗剂BX471。细胞培养至第5天进行TRAP染色,提取不同条件作用后RAW264.7细胞的总RNA,采用Real-time PCR检测CCR1及破骨细胞标志基因cathepsin K、TRAP mRNA的表达,使用细胞免疫荧光和Western blot检测不同时期RAW264.7细胞中CCR1及Cathepsin K蛋白的表达量。结果加入A549细胞条件培养基的RAW264.7细胞TRAP阳性细胞显著增多,条件培养基明显上调RAW264.7的CCR1、Cathepsin K、TRAP的表达且呈浓度依赖,CCR1特异性拮抗剂BX471可抑制肺癌分泌因子对破骨细胞的活化。结论肺癌细胞分泌因子可促进破骨前体细胞向破骨细胞分化,CCR1参与了肺癌细胞分泌因子对破骨细胞的活化过程。
Objective To establish an indirect co-culture model of lung cancer cells with monocytes / macrophages to observe the effect of CCR1, a precursor of osteoclasts (RAW264.7), on osteoclastogenesis induced by lung cancer cell conditioned media. Methods Lung cancer cell A549 conditioned medium was collected. The conditioned medium was added into RAW264.7 medium at different concentrations and co-cultured with or without CCR1-specific antagonist BX471 in the co-culture system. TRAP staining was performed on the fifth day after cell culture and the total RNA was extracted from RAW264.7 cells under different conditions. Real-time PCR was used to detect the expression of CCR1 and cathepsin K and TRAP mRNA. The expression of TRAP mRNA, blot was used to detect the expression of CCR1 and Cathepsin K in RAW264.7 cells. Results The number of TRAP-positive cells in RAW264.7 cells was significantly increased in A549 cells. The conditioned media significantly increased the expression of CCR1, Cathepsin K and TRAP in RAW264.7 cells in a concentration-dependent manner. The CCR1-specific antagonist BX471 inhibited the secretion of TRAP Factor on osteoclast activation. Conclusions Secretion factors of lung cancer cells can promote the differentiation of osteoclast precursors into osteoclasts. CCR1 is involved in the activation of osteoclasts by lung cancer cell secretory factors.