Establishment The Animal Model Of Bone Metastasis Of Breast Cancer And Analysis The Molecular Mechanisms Of Metastasis

OBJECTIVE:to establish animal model of bone metastasis of breast cancer using human breast cancer cell lines MDA-MB-231 transfected with GFP via left ventricle of heart and research on the differential expression of breast cancer-related genes by gene chips technology and further explore the molecular mechanism of bone metastasis of breast cancer.METHOD:We transfected the vector expression GFP into MDA-MB-231 cells at logarithmic growth phase using liposome transfection method and detected that they stable expressed GFP under fluorescence microscopy.Then drawing growth curve in vitro of the two groups cells before and after transfection,a statistical comparison was done to determine whether the impact of the transfected cell activity and tumorigenicity.Next step we injected MDA-MB-231/GFP cells into nu/nu nude mice via the left ventricular.Forty-five days later after injection,PET-CT scan was done to detect whether bone metastasis occurred and then all the animals were sacrificed.Metastasic organization were taken about sized 1.5 cm×0.5cm×0.3cm to do frozen section and observed the expression of green fluorescent protein.The remained were preserved in 10%neutral formalin and the metastatic organization were embedded by paraffin and then got the sections.During the anatomy process,the skeletal system of nude mice should be complete and X-ray examination can be taken clearly.Primary MDA-MB-231/GFP cells were collected and lysised in Trizol completely.Metastasic tissues were preserved in RNAlater.The two samples were sent to biochip company in shanghai to do the commercial analysis.According to the gene clip,CXCR4 and NF-KB which had significant difference in bone matastasis tissue were choosed to do the further study. Immunohistochemical detections were done to observe the expressions of CXCR4 and NF-KB in the Primary MDA-MB-231/GFP cells and bone metastasic tissues. RESULT:Using liposome transfection method to transfect the vector expressioning GFP into MDA-MB-231 cells,then in the present of drug G418,the cells can be selected for stable transformation.MDA-MB-231/GFP cells got a higher proportion after selected by flow cytometry.The cell growth curves were delineated to analyze the cells proliferating dynamic values.And the cell growth curves the two groups' cells before and after transfection were basically consistent,and we could come to the conclusion that the activity and tumorigenicity of MDA-MB-231/GFP had not been changed by transfection.There were sixteen nude mice in the experimental group,and the metastasis of ribs and chest were found in ten of them,and lung metastasis was found in four of them,and no metastasis was found in two of them.Bone lesions were observed in PET-CT and X-ray images.Different sizes of white round nodule were found in libs,chest and other sites during the course of anatomy.Frozen sections showed that the green fluorescent protein exited in the tissue we supplied.Pathological biopsy results suggested that tumor cell infiltrated.The analyses of differential genes indicated that there were 176 genes which were up-regulation and 298 genes down-regulation.Compared with primary cells,the expression of CXCR4 in bone metastasic tissues was up-regulation significantly.Imunohistochemistry results showed that CXCR4 and NF-KB expressions were weaker in the cells of climbing flake of MDA-MB-231/GFP and only part of the cytoplasm were was positive,which was stronger in the metastatic organizations;almost all of the cytoplasm was full of brown-color.CONCLUSION:The integration and expression of GFP have no influence on the biological characteristics of human MDA-MB-231 cells and can be used on the further study of the mechanisms of breast cancer bone metastases and treatment as a report gene.Left ventricular injection could imitate the course of development and metastasis of breast cancer bone metastasis in the great degree.It is credible and feasible to establish the animal model of bone metastasis of breast cancer.The analyses of differential genes indicated that there were 176 genes which were up-regulation and 298 genes down-regulation.The expression of chemokine receptor(CXCR4)and NF-KB in bone metastatic organization of breast cancer was significantly higher than that of the primary cells.The results showed that CXCR4 and NF-KB could be the most important chemokine which can lead to bone metastasis of breast cancer and CXCR4 and NF-KB are likely to be new targets for the treatment of bone metastasis in breast cancer.