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.

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.

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.

Objective To discuss the diagnosis and treatment of insulinoma.Methods The clinical data of 30 patients with insulinoma were reviewed.Results All patients had Whipple′s triad.Accurate preoperative localization rate of B ultrasonography, CT and MRI was 34.8 %(8/23),58.3 %(7/12) and 71.4 %(5/7),respectively. Localization rate of intraoperative ultrasonography (IOUS) was 87.5 %(7/8). The tumors were single in 27 cases,and multiple in 3 cases. In the location of single tumor,8 of them were in the head,7 in the body, and 12 in the tail; while for multiple tumors, 2 tumors were both located in the body in 1 patient,and 2 tumors were separately located in the body and tail respectively in 2 patients. Local enucleation was performed in 21 cases, distal pancreatectomy in 6 cases,distal pancreatectomy plus splenectomy in 2 cases,and duodenopancreatectomy in 1 case. The tumor was benign in 29 cases, and malignant in 1 case.Pancreatic fistula developed after operation in 4 cases, and in all cases, it healed after drainage. All patients had no symptoms of hypoglycemia after operation. At follow-up visit in 27 cases, 1 case of benign tumor recurred 4 years after operation, and was cured by resection of the pancreas body with tumor; the malignant tumor case, recurred and died of metastasis of abdominal cavity 3 years after operation.Conclusions Whipple′s triad, and the ratio of immunoreactive insulin to blood glucose (IRI/G)are the bases for qualitative diagnosis of insulinoma. Meticulously palpating the gland combined with IOUS during operation is the most effective method for accurate tumor localization. Enucleation is the main mode of surgical treatment of insulinoma.

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

OBJECTIVETo 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.

METHODSRecombinant 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.

RESULTSBoth 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.

CONCLUSIONSThe malignant phenotype of OS732 is inhibited and GJIC is increased by transduction of N-terminal truncated Rb gene.

Bone Neoplasms ; Cell Communication ; Cell Line ; Genes, Retinoblastoma ; Humans ; Osteosarcoma ; Tumor Cells, Cultured

Objectives To detect the nm23 expression in osteosarcoma, analyze the correlation between nm23 expression and early metastasis and to study the relation between nm23 expression with tumor differentiation and cell proliferation, as defined by Ki-67 labeling.Methods nm23 oncoprotein expression was measured by immunohistochemical staining using formalin fixed, paraffin embedded sections of 39 surgically resected osteosarcomas and was compared with Ki-67 expression, histological findings and early metastasis. In each sample, the grading of immunoreactivity was recorded as 0, 1+, 2+ according to the percentage of nm23 and Ki-67 labeling cells respectively.Results The cases with more than 25% positive immunoreactivity of nm23 protein were detected in 48.7% of the total cases. There was no statistical difference between nm23 expression and early metastasis, but the cases with nm23 expression appeared to have a trend to get early metastasis. nm23 expression was significantly correlated with Ki-67 expression, which was associated with differentiation of the tumor cells.Conclusion The role of nm23 as a tumor metastasis suppressor in osteosarcoma is less prominent. Both nm23 and Ki-67 may help predict the possibility of early metastasis in human osteosarcomas.