Effect Of Recombinant Replication-defective Adenovirus Vector Mediated Human IL-24/MDA-7 Gene Transfection On Pancreatic Carcinoma Growth In Vitro And In Vivo

Objective:This study was conducted (1) to construct a recombinant adenoviral vector carrying IL-24 gene(Ad-IL-24);(2) to detect the change of the human pancreatic carcinoma cell patu8988 infected with Ad-IL-24 and elucidate the molecular mechanism of Ad-IL-24 in inhibiting the growth of pancreatic carcinoma(PC);(3)to analyze the variations of the surface phenotype and immuno-status of dendritic cells that they had been IL-24 gene modified;(4) to observe the growth of PC in vivo,and demonstrat the effect of Ad-IL-24 in treatment of PC.Methods:Total RNA was extracted from PBMC, and then a 621bp fragment at the 5' end of human IL-24 cDNA was synthesized by reverse-transcription polymerase chain reaction (RT-PCR) and was reversely inserted into the multiclone site (MCS) of the shuttle plasmid pAdTrack-CMV,with the resultant plasmid and the backbone plasmid pAdEasy-l,the homologous recombination took place in the E.coli DH5a and the recombinant adenoviral plasmid carrying the IL-24 gene was constructed was generated. The adenoviruses(Ad-IL-24) were packaged and amplified in the HEK 293 ce1ls. Ad-IL-24 infected the human pancreatic carcinoma cell line patu8988. Then the production of IL-24 in the human pancreatic carcinoma patu8988 cells was detected with Western Blot Analysis,ELISA and FCM. According to Inaba'smethods,bone marrow-derived Dcs were induced. The surface molecules of DCs are tested by FCM. Following the Ad-IL-24-infected cells was subcutaneously inoculated in nude mice, and injected intratumorally into pre-existing tumors.After four weeks,the tumor tissue was detected with IHC and RT-PCR.Results:The recombinant adenovirus vector carrying IL-24 was constructed Successfully,fluorescence the strong green fluorescence was microscopy. The viral titer was observed in HEK 293 cells under a fluorescence microscopy.The viral titer was 5×1010 pfu/ml. Compared with PBS or Ad-GFP-infected patu8988 cells, infection of patu8988 cells with Ad-IL-24 significantly reduced the growth of human pancreatic carcinoma patu8988 cells,and the tumor volume resulted in reduction in nude mice.In addition,direct intratumoral injection of Ad-IL-24 into pre-existing tumors significantly impaired the further expansion of the tumor mass and resulted in a reduction in tumor.Ad-IL-24 therapy resulted in a reduction in tumor vessel density.In vitro,Ad-IL-24 induce the phenotypic and functional maturation of DC. Beside these results, the growth rate of PC significantly reduced and the survivals prolonged in the Ad-IL-24-infected nude rate modelsConclusion:(1)The recombinant adenovirus with IL-24 can effectively inhibit cells growth and promote the apoptosis of human pancreatic carcinoma patu8988 cells. (2)Ad-IL-24 induce the phenotypic and functional maturation of DC.(3)Ad-IL-24 can effectively prolong survivals and attenuate angiogenesis of pancreatic carcinoma in vivo models and demonstrat a therapeutic potential for human pancreatic carcinoma.