| Breast cancer is one of the most common malignant tumors attacking women .The morbidity and mortality are increased with years and the mortality is highest in female cancers. According to the statistics, more than 30 percent of breast cancer patients die from tumor metastasis. Even if the primary cancer is totally excised by operation, metastasis is a key factor which can influence on survival of breast cancer patients. Tumor metastasis is a comprehensive process involving multivariate and multiple stages. Each cancer type has its own preferred metastasis sites. While there has been considerable progress in identifying genes that promote the metastasis of cancer cells, little is known about the genes that enable cancer cells to plant, survive, and proliferate at metastasis sites. It has been indicated that many tumor cells may express certain chemokines and chemokine receptors definitely, and there exists disregulation of signaling pathways which consist of chemokines and their receptors. It is confirmed that chemokines and their receptors play important roles in providing the migration signal for inflammatory cells and then activating them to initiate directional migration to specific sites accurately .These may suggest that they participate in tumor development, malignant transformation, trafficking and metastasis with the metastatic pattern similar to the infiltration of inflammatory cells.CXC type chemokine receptor 4(CXCR4) is a member of G-protein coupled recepors family. CXCR4/CXCL12 axis formed with CXCR4 and its ligand SDF-1 can mediate many effects on matrix and tumor cells including activation of the Ras/MAPK signal transduction pathway, actin polymerization, cytoskeletal reorganization, pseudopodia formation and subsequently promoting cell migration and invasive responses. To elucidate the relationship between CXCR4 and breast cancer, and then to evaluated the probability of CXCR4 as a molecular marker of breast carcinoma progression and a medication therapy target, this work gained the fusion protein CXCR4 and its polyclonal antibody after cxcr4 cloning and expression in Ecoli BL21. In this study, the primer was designed according to cxcr4 sequence deposited in GenBank (NO. NM003467), and the total RNA was extrated from normal human PBMC and used as a template for RT-PCR. To construct recombinant plasmid pMD-cxcr4, the 1059bp fragments containing the whole length coding region of CXCR4 were inserted into pMD18-T vector when it was purificated. After antibiotic selection and restrictive enzyme digestion, it was proved that the recombinant plasmid pMD-cxcr4 had been constructed successfully. The target gene contained in recombinant plasmid pMD-cxcr4 was sequenced and compared with the full lenth coding region of CXCR4 deposited in GenBank (NO. NM003467). It showed that only two bases were different, but each of the encoding amino acid was consistent. After the recombinant plasmid pMD-cxcr4 being digested with EcoR I/HindⅢ, the cxcr4 fragment was extracted and subcloned into pET-28a(+) to construct expression recombinant plasmid pET-cxcr4 and then transformed into Escherichia coli BL21. The expressed protein induced by IPTG was detected by SDS-PAGE and a band about 43kD was produced, which was corresponded with that of expected. The purified fusion protein was used to immunize New Zealand rabbit to prepare anti-CXCR4 immune serum. The antibody liter was detected by non-competitive ELISA and the valence was 1:8000. The obtaining of CXCR4 fusion protein and its antibody were benefit to further research on the relationship between CXCR4 and breast cancer.To investigate the association of CXCR4 expression with carcinogenesis and development of malignant breast tumor, immunohistochemistry for CXCR4 was performed on specimens of 45 cases pathological tissues.The expression of CXCR4 and cell localization in breast carcinoma were analyzed at protein level. CXCR4 staining was positive in 25 cases of breast cancer, especially showed strong positive reaction in cancer nests. Immuno-localization assay indicated that CXCR4 was expressed in cellular membrane, cytoplasm and extracellular matrix. In the specimens of 20 cases breast cancer, CXCR4 staining was negative in normal tissue around cancer. The benign breast hyperplasia eases appeared to be negative or weak positive staining for CXCR4 immunohistochemistry. It was explanated by statistic analysis on diagnostic ease that strong positive was mostly seen in the cases of breast cancer matastasis, as stated below: 13 eases were++～+++; other 12 cases showed +～+++ in CXCR4 dyeing intensity, no significant differences. Based on the immunohistochemistry results, serologically tests were performed on breast cancer patients to detect CXCR4 by competitive ELISA. The statistical results suggested that the comparative content of antigen CXCR4 in breast cacinoma eases, benign breast hyperplasia cases and normal persons was 67.15±8.37, 18.25±5.56, 9.793±2.62, respectively. Serum content of CXCR4 in breast cancer patients was 6.9 folds to normal persons and 3.7 folds to benign breast hyperplasia cases. This study was further confirmed that the expression of CXCR4 was closely associated with breast carcinoma and can be used as a molecular marker for it.
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