Construction of pIRES2-HBSP-EGFP vector and its expression in breast cancer cells / 中国组织工程研究

ABSTRACT

BACKGROUND: Bone sialoprotein (BSP) gene is expressed in human breast cancer cells, in which bone metastasis occurs easily outside the mineralized tissue. Clinical observation shows that the expression level of BSP of breast cancer cells at bone metastasis is higher that at the primary site;therefore, BSP may be closely related to tumor specific bone metastasis.The study on breast cancer bone metastasis can provide new drug target for clinical prevention and treatment.OBJECTIVE: To establish breast cancer cell strains of BSP with stable expression and observe the effect of BSP in the whole process of breast cancer bone metastasis.DESIGN: Controlled experiment.SETTING: College of Biological Sciences and Engineering, South China University of Science and Technology; Medical Experiment Center,Guangzhou General Hospital of Guangzhou Military Area Command of Chinese PLA.MATERIALS This experiment was conducted in the Medical Experimental Center,Guangzhou General Hospital of Guangzhou Military Area Command of Chinese PLA,betweer November 2003 and March 2004..pIRES2-EGFP vector (5.3 kb) was purchased from BD Biosciences Clontech Inc.; E.Coli.Top10, pB-hBSP plasmid containing the coding region of hbsp, and human breast carcinoma cells, MDA-MB-231BR that was specifically transferred to brain and MDA -MB-231BO that was specifically transferred to bone.METHODS: hbsp gene was subcloned from pB-hBSP vector by PCR. Bg1Ⅱ and Pst Ⅰ restriction enzyme sites were inserted at 5' and 3' ends, orientation cloned to eukaryon expression vector pIRES2-EGFP, and constructed recombinant vector pIRES2-EGFP. The constructed recombinant vector was transfected into MDA-MB-231BR that was specifically transferred to brain and MDA-MB-231BO that was specifically transferred to bone.MAIN OUTCOME MEASURES: Construction of pIRES2-hBSP-EGFP recombinant expression vector; recombinant expression vector pIRES2-hBSP-EGFP transfecting breast cancer cells.Breast cancer strains specific in bone metastasis and brain metastasis were successfully transfected. The fluorescence labeling could be observed under the fluorescence microscope, and BSP had corresponding expression.CONCLUSION: The successful construction and transfection of pIRES2hBSP-EGFP of eukaryon expression vector would lay foundation for further study on the role of BSP in breast cancer metastasizing to bone in vivo or in vitro.