Transfection Of Osteosarcama Cells With Sense And Antisense Human Bone Sialoprotein Eukaryotic Expression Vectorinfluence On Behavior Of Adhesion And Invasion Of Osteosarcoma

Objective To construct sense and antisense human bone sialoprotein(BSP) eukaryotic expression vector for further studying the effective of hBSP , The sense and antisense hBSP eukaryotic expression vector was transfected into MG-63 cells and its expression detected in human osteosarcoma cell line MG-63 cells.To preliminarily study its biological function in adhesion and invasion of osteosarcoma. Methods The open reading frame of Human BSP was amplified by RT-PCR method from human periosteum and inserted into pUCm-T vector to form cloning plasmid.Then the ragment of target was subcloned into eukaryotic expression vector pIRES2-EGFP in orientation thought utilized the difference restriction enzyme cut site of both end of the primers.The recombinant plasmid was identified by restriction enzyme digestion analysis and sequencing analysis after bacterial transformation, amplification and purification. The sense and antisense hBSP eukaryotic expression vector was transfected into MG-63 cells with lipofectamine. The expression of BSP mRNA on the cells was measured by RT-PCR, The expression of BSP on the cells was measured by Western blotting. MTT assay was used to test transfected cells adhesion on extracellular matrix protein. Transwell-chamber method was used to evaluate influence of BSP expression on osteosarcoma cell invasion. Monolayer wound healing assay was used to measure osteosarcoma cell migrationResults The open reading frame of Human BSP was successfully amplified from human periosteum The sense and antisense hBSP eukaryotic vestor was successfully constructed and transfected.Compared with control cells,expression of BSP increased in the cells by sense hBSP eukaryotic vector transfected,adherence ratio of these cells is raise (P<0.05),invasive power of these cells is enhance(P<0.05) and migration rate of these cells is increase(P<0.05). Expression of BSP decreased in the cells by antisense hBSP eukaryotic vector transfected, adherence ratio of these cells is degrade (P<0.05) , the ability of invasive of these cells is been inhabited(P<0.05) and migration rate of these cells is decrease(P<0.05).. Conclusion Osteosarcoma cells can express BSP. Up-expression of BSP can enhance adherenc,invasive and migration of osteosarcoma cell, antisense hBSP eukaryotic vector transfected have inhibitory effects on many aspects or steps associated with metastasis of osteosarcoma, The change of BSP expression may play an important role in osteosarcoma adhesion and migration.