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<p><b>OBJECTIVE</b>To investigate the effect of growth suppression and gap junctional intercellular communication (GJIC) on osteosarcoma cell line OS732 transduced with the exogenous N-terminal truncated retinoblastoma (Rb) gene.</p><p><b>METHODS</b>Recombinant eukaryotic expression plasmid harbouring 1.65 kb Rb gene was constructed and transduced into OS732 cells. Rb mRNA expression was detected by RT-PCR and Northern blot. The cell count, cell cycle analysis and soft agar colony formation test were used to observe growth features. RT-PCR and Lucifer yellow dye transfer were carried out to measure GJIC.</p><p><b>RESULTS</b>Both exogenous and endogenous Rb gene could be detected in OS732 cell transduced with the N-terminal truncated Rb gene. Compared with the parental cells, change in morphology of Rb-transduced cells were found; growth rate decreased; amount of colony formation reduced; cell cycle arrested at G(0)/G(1) checkpoint; Connexin43 expression and GJIC enhanced.</p><p><b>CONCLUSIONS</b>The malignant phenotype of OS732 is inhibited and GJIC is increased by transduction of N-terminal truncated Rb gene.</p>
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Humanos , Neoplasias Ósseas , Comunicação Celular , Linhagem Celular , Genes do Retinoblastoma , Osteossarcoma , Células Tumorais CultivadasRESUMO
Objective To explore an alternative strategy of ganciclovir-mediated cytotoxicity and study the mechanism of enhancing bystander effect of HSV-TK/GCV system by combination gene transfer of Rb gene and HSV-TK gene.Methods Recombinant eukaryotic expression plasmid pcDNA3.1-Rb harbour ing1.65kb of Rb gene was constructed.The pcDNA3.1-Rb and the plasmids tgCMV/HyTK car-ry ing HSV-TK gene were transfected respectively or co-transfected into the osteosarcoma cell line OS732by lipofection using DOTAP reagent.The mRNA expression of Rb gene and HSV-TK gene were detected by RT-PCR and Northern blot.Cell counting,cell cycle analysis and soft agar colony formation test were adopted to observe cell growth features.The expression of gap junction Connexin43gene was performed by RT -PCR and Western blot.The direct confirmation of gap junction intercellular commu ni cation was demonstrated by Lucifer yellow dye transfer.Cell sensitivity to GCV and″bystander effect″of HSV-TK/GCV system were measured by MTT assay.Results The eukaryotic expression plasmid pcD NA3.1-Rb was constructed successfully.The transfected cell lines OS732TK,OS732Rb and OS732RbTK were har-vested.mRNA expression of HSV-TK gene was demonstrated in OS732TK and OS732RbTK cells.Both exogenous and endogenous Rb gene expression could be detected in OS732Rb and OS732RbTK cells si-multa neously.Compared with parental cell OS732,OS732Rb and OS732RbTK cells changed their mor -phology and decreased the growth rate;the ability of soft agar colony formation reduced and the cell cycles were arrested at G 0 G 1 checkpoint.The Connexin43expression and gap junction in tercellular communica-tion en hanced in OS732Rb cell.GCV was of toxicity to only TK -positive cells,OS732TK and OS732RbTK,in a concentration dependent manner,the mixed coculture experiments of OS732and OS732Rb with the TK-negative cell,both showed sensitive to GCV,but the survival rate of OS732Rb cell was significantly lower than OS732cell under the same condition.Conclusion Coordinate ex pression of Rb andHSV-TK gene conferred a more po tent bystander effect by enhancing gap junction intercellular communica-tion and in hibiting prolifera tion.
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AIM: To investigate killing effect of herpes simplex virus thymidine kinase/ganciclovior system(HSV-TK/GCV) on osteosarcoma cell and its mechanisms.METHODS: Recombinant retroviral vector (DORHyTK) containing hygromycin phosphotransferase-thymidine kinase fusion gene(HyTK) was constructed and introduced into human osteosarcoma cell line OS732 with DOTAP. DNA and total RNA extracted from HyTK expressing cells (OS732TK) were tested by PCR and RNA dot blot analysis. A chemosensitivity of OS732TK cells to GCV and “bystander effect” were measured by means of MTT colorimetric assay. Hoeschst 332258 staining and flow cytometric analysis were performed for mechanism of HSV-TK/GCV system gene therapy. RESULTS: DORHyTK was constructed successfully; the HyTK gene existed and expressed in OS732TK cells; All OS732TK cells were killed after 5 days of exposure to 5 mg/L GCV. The “bystander effect” was observed in both a high population and a low population, but the former was stronger than the latter. Hoeschst 332258 staining revealed the characteristic hallmarks of apoptosis, however necrosis also existed. Flow cytometric analysis of DNA content showed a G0-G1 phase blockade. CONCLUSION: HSV-TK/GCV gene therapy system showed its strong killing effects on osteosarcoma cells OS732; The phenomenon of “bystander effect” was also very apparent. GCV exposure induced both necrotic and apoptotic death in HSV-TK expressing cells, and perturbed the cell cycle. HSV-TK/GCV gene therapy system may provide a new therapeutic approach for treatment of osteosarcoma.
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Purpose To observe the killing effect of HSV-TK/GCV system on osteosarcoma cells. Methods The eukaryotic exprssoin vector (tgCMV-HyTK) containing HSV-TK gene was transduced into human osteosarcoma cell lines SAOS-2 by DOTAP. Total RNA extracted from HSV-TK expressing cells (SAOS-2TK) were tested by RNA dot blot analysis. A chemosensitivity of SAOS-2TK cells to GCV and “bystander effect” were measured by means of MTT colrimetric assay. Results HSV-TK gene was expressed in SAOS-2TK cells. SAOS-2TK cells were susceptible to the cytotoxicity of GCV. A meaningful “bystander effect” was demonstrated. Conclusion HSV-TK/GCV system may kill selectively the human osteosarcoma cell line SAOS-2.
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Objective To study the biological functions of growth factors in the process of osteoblasts allografting and its acceleration in fracture healing in osteoporosis rats. Methods The fracture model of rat osteoporosis was established and then cell grafting was performed. Immunnohistochemistry and in situ hybridization were utilized to examine the expression of bFGF and bFGF mRNA during the process of fracture healing, the image was also analyzed. Results In the experimental group, bFGF positive cells were seen after 7 days after cell grafting, and peaked in chondrocytes about 14 day (384 65?8 60) after grafting. bFGF began declining after 21 days (286 24?2 30) and disappeared after 56 days. However in the control group, obvious high quanty was not seen and being only 125 33?4 50 even peaked at 21st day. Conclusions bFGF not only plays a very important role in bone formation but also enhances circulation formation in the fracture areas. It also accelerates the fracture healing in osteoporosis rats.
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AIM:To study the expression of metastasis suppressor gene KAI1 mRNA in osteosarcoma tissue and osteosarcoma cell lines,and the relationship between it and the biological behavior of the tumor cells.METHODS:RT-PCR was used to detect KAI1 mRNA in 18 cases of resected fresh osteosarcoma samples and three cultured osteosarcoma cell lines.The proliferative rate,the adhesive and invasive abilities of the 3 cell lines were detected.The results were treated by analysis system of images and analyzed with t test.RESULTS:The relative amount of KAI1 mRNA in osteosarcomas with lung metastasis was 0.80?0.50,while that was 1.48?0.64 in osteosarcomas without lung metastasis,the former was significantly lower than the latter(P
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AIM: To investigate killing effect of herpes simplex virus thymidine kinase/ganciclovior system(HSV-TK/GCV) on osteosarcoma cell and its mechanisms.METHODS: Recombinant retroviral vector (DORHyTK) containing hygromycin phosphotransferase-thymidine kinase fusion gene(HyTK) was constructed and introduced into human osteosarcoma cell line OS732 with DOTAP. DNA and total RNA extracted from HyTK expressing cells (OS732TK) were tested by PCR and RNA dot blot analysis. A chemosensitivity of OS732TK cells to GCV and "bystander effect" were measured by means of MTT colorimetric assay. Hoeschst 332258 staining and flow cytometric analysis were performed for mechanism of HSV-TK/GCV system gene therapy. RESULTS: DORHyTK was constructed successfully; the HyTK gene existed and expressed in OS732TK cells; All OS732TK cells were killed after 5 days of exposure to 5 mg/L GCV. The "bystander effect" was observed in both a high population and a low population, but the former was stronger than the latter. Hoeschst 332258 staining revealed the characteristic hallmarks of apoptosis, however necrosis also existed. Flow cytometric analysis of DNA content showed a G 0-G 1 phase blockade. CONCLUSION: HSV-TK/GCV gene therapy system showed its strong killing effects on osteosarcoma cells OS732; The phenomenon of "bystander effect" was also very apparent. GCV exposure induced both necrotic and apoptotic death in HSV-TK expressing cells, and perturbed the cell cycle. HSV-TK/GCV gene therapy system may provide a new therapeutic approach for treatment of osteosarcoma.
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Bone morphogenetic protein-1(BMP-1) and its related molecules are members of metalloendoproteinase astacin family, including BMP-1, mTLD, mTLL-1 and mTLL-2. Even though all of them lack of the ability to induce bone or cartilage formation directly, they play key roles in numerable activities in ECM from embryo to adult, then affect the procedure and the result of osteogenesis and bone remodeling directly or indirectly. They are critical in maturation and deposition of some major collagen types, and in regulating the signaling of some growth factors in TGF-? superfamily by degradation of TGF-? inhibitor such as Chordin. The investigations about tissue distribution of BMP-1 and its related proteinases and also gene knock-out studies strongly indicate that they play key roles in osteogenesis and bone development.
