The Establishment And Identification Of Human Osteosarcoma Cell Lines Stably Expressing Wild-type P53 Gene And Tk1 Gene

The establishment and identification of human osteosarcoma cell lines Stably expressing wild-type p53 gene and tk1 geneObjectiveTo establish human osteosarcoma cells(U-2 OS) that stably express exogenetic wild-type p53 and tk1 gene, providing model establishment and identification method in vitro for further study of wild-type p53 gene's synergism in osteosarcoma bystander effect.MethodsUsing the molecular biology gene cloning technique, inserted human wild-type p53, tk1 cDNA fragments respectively into the expression vector pIRES-EGFP, obtained the recombinant plasmids pIRES-EGFP-p53 and pIRES-EGFP-tk1. Through PCR, enzyme electrophoresis and other methods, carryed on the identification of plasmids; Then the recombinant plasmids were transformed into E.coli BL21 cells, the transformed colonies were amplified by PCR, and the plasmid DNA was inducted into U-2 OS cells by liposome-mediated transfection method. Under fluorescence microscopy, the osteosarcoma cell lines stably transfection was obtained by manual Screening.Results1.Successfully constructed recombinant plasmids pIRES-EGFP-p53 and pIRES-EGFP-tk1 containing wild-type p53, tk1 cDNA fragments. Through PCR, enzyme electrophoresis and other methods to identify the size and position of plasmids, the result was consistent with the experimental design.2.Then inducted the recombinant plasmids into human osteosarcoma cell line U-2 OS cells, named U-2-p53 OS,U-2-tk1 OS and U-2-p53-tk1 OS. Meanwhile inducted the blank vector pIRES-EGFP into U-2 OS cells as the negative comparison, using the same method of transfection.ConclusionThe establishment of human osteosarcoma cell lines by gene transfer technology laid the foundation for further study of wild-type p53 gene in osteosarcoma bystander effect. The expression of tumor suppressor genes PTEN and p27 in the osteosarcoma tissues and its clinical significanceObjectiveTo explore the role of tumor suppressor genes PTEN, p27 in the development of osteosarcoma through the research about their expression in osteosarcoma tissues.MethodsPathological specimens and clinical data of 35 patients with osteosarcoma, 15 patients with fibrous dysplasia from 2003-01 to 2007-03 in our hospital were collected. SP immunohistochemistry method was used to detect expression of the PTEN and p27 in these pathological specimens. Then statistics analysis were carried on with clinical data. Applying SPSS13.0 statistical package, the comparison of two or more of enumeration data were carried out by Chi-square test or Fisher probabilities test, correlation of two factors were tested using Spearman rank correlation analysis. P value less than 0.05 was considered statistically significant.Results1.The positive rate of PTEN in osteosarcoma tissues was significantly lower than that in fibrous dysplasia tissues(χ~2=5.510, P=0.019). The expression level of PTEN in high differentiated osteosarcoma tissues was higher than that in low differentiated tissues(χ~2=9.121, P=0.003). Also, the loss or decreased expression of PTEN was significantly correlated with clinical stages(χ~2=8.117, P=0.004).2.The positive rate of p27 in osteosarcoma tissues was significantly lower than that in fibrous dysplasia tissues too(χ~2=6.731, P=0.009). There was a positive correlation between the expression of p27 with tissue differentiation(χ~2=3.998, P=0.046), while the loss or decreased expression of p27 was related to clinical stages and Price's grades(χ~2=15.04, P=0.001;χ~2=4.496, P=0.034).3.In osteosarcoma tissues, a positive correlation was not found between the expression of PTEN and p27(r=0.097, P=0.581).ConclusionPTEN and p27 are closely related to the occurrence of osteosarcoma. The detections of their expression may be helpful to early diagnose of osteosarcoma.