Construction And Characterization Of Replication Defective Adenovirus Vector Of Coding HSSTR2 Gene

Background & Objective: Somatostatin is a widely distributed peptide thatnegatively regulates multiple physiological functions. Somatostatin inhibits cell proliferation and growth hormone secretion through interaction with somatostatin receptors (SSTRs). Five SSTRs have been cloned in the human, among those SSTRs, the SSTR2 gene is found to be the most important SSTR subtype for tumors. Many studies have demonstrated tumor cell growth inhibition by somatostatin and somatostatin analogs in SSTR2-positive tumors. However, functional SSTR2 are lost in most pancreatic cancers. Loss of SSTR2 expression may lead to a deficiency in negative regulation of cell growth by somatostatin analogs. Accordingly, gene therapy may be a promising strategy. Treating pancreatic cancer by reintroducing SSTR2 gene back to the cancer cells may be a good way. Thus in the present study we established a SSTR2 replication disfigurement adenovirus carrier. In the course of gene therapy using SSTR2 in pancreatic cancer, Ad-SSTR2, the adenovirus carrier also function as the base of gene therapy.Methods: SSTR2 cDNA and LacZ cDNA were inserted into adenovirus shuttleplasmid pDC316, named pDC316-SSTR2 and pDC316-LacZ, respectively. pDC316-SSTR2 and pDC316-LacZ were cotransfected with rescue plasmid pBHGlox(A)El,3Cre into 293 cells by liposome mediating. Ad-SSTR2 and Ad-LacZ were generated by site-specific recombination and confirmed by PCR and immunoblotting. The course of Ad-SSTR2 expressing in the 293 cells was comfirmed by immunoblotting. Ad-SSTR2 and Ad-LacZ were propagated in 293 cells and purified. The titer of viral stock was determined by end-point dilution assay and the infective capability of recombinant adenovirus was measured by X-gal staining method. RT-PCR, Western blot and immunocytochemistry stain were employed to observe the...