The Effection Of Generation And Apoptosis Of Pancreatic Carcinoma Treated By A Retroviral Vector Containing Anti-Sense HTR Gene

Objective: To study the effection of generation and apoptosis of pancreatic carcinoma treated by a retroviral vector containing anti-sense hTR gene.Methods : we got the recombinant retroviral vector plasmid pLXSN-hTR, which carrying the retro-hTR gene. Sense and antisense telomerase RNA gene were transfected into packaging cell line PT67 by electroporation, and selected with G418 to get stable transfection cell lines, which could produce retrovirus, After the recombinant retrovirus supernatant was collected, the viral titer was determined with NIH3T3 cell line, we transfected Can-pan-2 cell with concentrated retroviral supernatant, and selected with G418 to get stable transfection cell lines, We except that the transformed pancreatic cancer cell mediated by the retrovirus vector could transit anti-sense telomerase RNA, which could combine with the normal telomerase RNA, then stop the expression of telomerase and induce the malignant tumor cell into apoptosis. We could confirm the expression of the aim gene in Can-pan-2 cell through PCR, TRAP-PCR-ELISA was used to determine the telomerase activity, immunofluorescence was adopted to determine reproductive activity of the infected cells, flow cytometry (FCM) were used to study the cell apoptosis.Results: The titer of sense and antisense recombinant retrovirus could reach 0.95×106 and 1.1×106 respectively, PCR demonstrated the telomerase RNA genes were successfully intergrated into the target cells' gene, TRAP-PCR-ELISA was used to determine the telomerase activity, The telomerase activity of Can-pan-2-hTR-BamHI was 0.67±0.07, compared with Can-pan-2-hTR-EcoRI (1.00±0.17) and Can-pan-2 (1.14±0.27), it was significantly inhibited; immunofluorescence show that the expression of PCNA in Can-pan-2 and Can-pan-2-hTR-EcoRI were 26.92±0.57 and 26.20±0.77, which were higher than Can-pan-2-hTR-BamHI ; and through FCM, we can see the apoptosis rate of different cells, the apoptosis rate in Can-pan-2-hTR-BamHI was 9.7 % , which was higher than Can-pan-2 ( 7.2 % ) and Can-pan-2-hTR-EcoRI (6.9%) .Conclusions: The transformed pancreatic cancer cell mediated by the retrovirus vector could transit anti-sense telomerase RNA, which could combine with the normal telomerase genes, then stop the expression of telomerase and induce the malignant tumor cell into apoptosis.