关键词: 基因, p53；mdm2基因；RNA干扰；前列腺肿瘤；质粒

Abstract:

Abstract:Objective
To construct the recombinant vectors that coexpress wild type p53(wt-p53) and siRNA-mdm2 for gene therapy in androgen independent prostate cancer .Methods The subcloning,T-Acloning,and PCR technique were used to construct the recombinant vectors,named pcDNA3.1-p53,siRNA-mdm2,p53/siRNA-mdm2, and siRNA-scramble .The recombinant expression vectors mentioned above were transfected into PC-3 cells,the expression levels of the mdm2 siRNA and p53 after transfection were detected by the semi-quantitative RT-PCR analysis and Western blotting analysis.MTT assay was used to detect the  proliferation inhibition  of PC-3 cells after transfection.Results The recombinant plasmids containing both wt-p53 and siRNA-mdm2 were successfully constructed,and confirmed by DNA sequence analysis.After transfected for 48 h, the expression of GFP was observed.The expression level of wt-p53 was increased,and the expression of mdm2 was decreased detected by RT-PCR and Western blotting.MTT assay showed that the inhibitory rate of  proliferation of PC-3 cells was 40.4% after transfection.Conclusion The recombinant plasmids containing both wt-p53 and siRNA-mdm2 are successfully constructed,they can remarkablely inhibit the proliferation of PC-3 cells.

Key words: genes,p53；mdm2 gene；RNA interference；prostate cancer；plasmids