CCR1参与肺癌分泌因子诱导破骨细胞生成

CCR1 participates in lung cancer cell-secreted factors induced osteoclastogenesis

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。

Lung cancer is frequently metastasized to bone resuhing in osteolytic lesions, but the mechanism still unclear. The aim of this study is to establish an indirect cocuhure model of lung cancer cells and monocytes/ giant maerophages, and observe the effect of lung cancer A549 CM on RAW264.7 cell in the absence and presence of the specific antagonist for CCR1. RAW264.7 medium was cocuhured with different concentration of A549 condition medium in the presence or absence of CCR1-sepercific antagonist BX471. Five days later, tartrate-resistant acid phosphatase staining was carry out to determine the osteoclast formation. At the same time, the CCR1, TRAP, Cathepsin K mRNA expression in RAW264.7 were studied via Real-time PCR, and the proteins of CCR1 and Cathepsin were detected by immunofluorescence and Western blot. We found that lung cancer A549 CM significantly stimulated the formation of osteoclasts in coculture system compared with normal control; the specific antagonist for CCR1 could significantly inhibit osteoclastogenesis in the presence of lung cancer A549 CM compared with the control. We concluded that lung cancer A549 CM can promote osteoclast differentiation and CCR1 involved lung cancer-induced osteoclast activation.