中文摘要：

目的：采用体外共培养技术模拟骨微环境,研究人骨形态发生蛋白9（bone morphogenetic protein9,BMP9）对人高骨转移性乳腺癌MDA-MB-231细胞迁移和凋亡的影响,并初步探讨其作用机制。方法：将重组腺病毒AdBMP9导入MDA-MB-231细胞中,与骨髓基质细胞HS-5在Transwell共培养体系中培养3d。然后,采用划痕愈合实验、Transwell小室和FCM法检测MDA-MB-231细胞迁移和凋亡的变化;RT-PCR法检测共培养的2种细胞中基质细胞衍生因子-1（stromal cell derived factor-1,SDF-1）、白细胞介素-6（interleukin6,IL-6）和单核细胞趋化蛋白-1（monocyte chemoattractant protein-1,MCP-1）等转移相关因子的水平;细胞免疫荧光法检测MDA-MB-231细胞中趋化因子受体CXCR4的表达;蛋白质印迹法检测2种细胞中SDF-1/CXCR4-PI3K信号通路关键分子的表达水平变化。结果：在模拟骨微环境的共培养体系中过表达BMP9后,乳腺癌MDA-MB-231细胞的迁移受到明显抑制（P〈0.05）,而肿瘤细胞凋亡增加（P〈0.001）。过表达BMP9后,骨髓基质HS-5细胞中转移相关因子SDF-1、IL-6和MCP-1表达显著下调（P〈0.05）,但MDA-MB-231细胞中SDF-1表达水平无明显变化,IL-6和MCP-1的表达也明显下调。另外,MDA-MB-231细胞中本身CXCR4阳性,BMP9过表达对MDA-MB-231细胞中CXCR4表达无明显影响,但可以下调其配体SDF-1在HS-5细胞中的表达水平,且MDA-MB-231细胞中磷酸化的Akt（phosphorylated Akt,p-Akt）水平也被下调（P〈0.05）。结论：模拟骨微环境的共培养体系中,BMP9过表达可以调节乳腺癌细胞与骨髓基质细胞的相互作用,抑制乳腺癌MDA-MB-231细胞的迁移,并促进其凋亡,且该抑制作用可能与SDF-1/CXCR4-PI3K信号通路有关。

英文摘要：

Objective： To investigate the effects of human BMP9 （bone morphogenetic protein 9） on migration and apoptosis of human breast cancer MDA-MB-231 cells in simulated bone microenvironment,and to explore its mechanism.Methods： MDA-MB-231 cells were infected with recombinant adenovirus AdBMP9 or AdGFP,then the cells were co-cultured with HS-5 bone marrow stromal cells for 3 d.The expression level of BMP9 was detected by RT （reverse transcription）-PCR and Western blotting.Woundhealing test,Transwell migration test and FCM （flow cytometry） were used to determine the changes of migration ability and apoptosis of MDA-MB-231 cells.Metastasis-related factors,including SDF-1 （stromal cell-derived factor-1）,IL-6 （interleukin-6） and MCP-1 （monocyte chemoattractant protein-1） in MDAMB-231 and HS-5 cells,were screened by RT-PCR.Immunofluorescence method was used to detect the expression of CXCR4 in MDA-MB-231 cells.Western blotting was carried out to detect the key molecules of SDF-1/CXCR4-PI3K signaling pathway in MDA-MB-231 and HS-5 cells.Results： In simulated bonemicroenvironment of co-culture system,BMP9 over-expression could significantly inhibit the migration （P〈0.05） and promote apoptosis of MDA-MB-231 cells （P〈0.001）.Compared with the control groups,BMP9 over-expression decreased the expession levels of IL-6 and MCP-1 in HS-5 and MDA-MB-231 cells （P〈0.05）.However,the level of SDF-1 was decreased only in HS-5 cells,but not changed in MDAMB-231 cells.The MDA-MB-231 cells expressed CXCR4 themselves,and BMP9 over-expression could not change the expression level of CXCR4.The levels of SDF-1 in HS-5 cells and p-Akt （phosphorylated Akt） in MDA-MB-231 cells were down-regulated after BMP9 over-expression （P〈0.05）.Conclusion： BMP9 can inhibit the migration and promote the apoptosis of breast cancer MDA-MB-231 cells in simulated bone microenvironment by regulating the cross-talk between MDA-MB-231 cells and HS-5 bone marrow mesenchymal cells.SDF-1/CXCR4-PI3K pathway may be