Experimental Studies On Therapy For Lung Carcinoma By PTEN Gene Mediated By Adenovirus Vector

Lung caner is one of the most common malignancies whose incidence is increasing more rapidly. The lack of effective screening procedures means the majority of patients present in later stages in which tumors are often resistant to even multimodal therapy. Thus, there is an urgent need for new approaches to lung cancer treatment. With the quick development of molecular biology, many advancements have been made in gene therapy. The tumor suppressor genes play a critical role in tumorigenesis and tumor development that they are often used as targets in gene therapy. PTEN is a tumor suppressor gene, which frequently mutated or lost in lung cancer, especially in non-small lung cancer. By now, the mechanisms of PTEN in the original occurrence, progression and development of lung carcinoma have not clear throughly and the researches about the relationship with PTEN and lung carcinoma are few.PARTâ… AIM: To construct recombinant adenovirus vector carrying PTEN (Ad-PTEN) Methods: After amplication of PTEN by primer VT415+VT416, PCR-PTEN and PSU-CMV were cut off with endonuclease EcoR I and Sal I and then the cDNA fragment obtained from digestion was subcloned into pShuttle-CMV to create a new shuttle vector pShuttle-CMV-PTEN . The newly-recombinated pSUCMV-PTEN was tested by restriction endonulcease digestions. Then plasmid PSUCMV-PTEN and Pbhge3 were transfected by Lipofectamine2000 into adenovirus packaging cell, HEK293. The recombinant adenovirus Ad-PTEN, a non-proliferatable recombinant adenovirus, was obtained from the lysates of HEK293 pellet by repeat froze-melt procedure and the titer of it was estimated.Results : The cloning sites and directions of insert of PTEN in the structure of pShuttle-CMV-PTEN were exactly confirmed by digestion of two restrictive endonucleases, followed by DNA sequence analysis. The recombinant adenovirus plasmid PSUCMV-PTEN and Pbhge3 were transfected by Lipofectamine2000 into adenovirus packaging cell, HEK293.After 9~14 days, while the cytopathic effects (CPE), such as cell...