Construction Of Adenovirus Vector Containing Human IL-12Gene And Its Anti-tumor Mechanism

Purpose: To compare the efficiency of3different transfectionmethods to4different cells for determining the suitable transfectionmethod for various cells. To construct a recombinant adenovirus expressionvector carrying human IL-12(hIL-12) gene, and investigate its effects onthe proliferation and TGF-β expression of Hep3B cells. To infect themonocytes by adenovirus carrying IL-12and observe the formation oftumor-associated macrophages (TAM) in the tumor microenvironment.Methods: Human K562, human monocytes, mouse adipocyte3T3-L1and mouse Hep A1-6were transfected by adenovirus Ad5-GFP,Ad5F35-GFP and plasmid pEGFP-N1(with Lipofectamine2000),respectively. Green fluorescence was detected under the fluorescencemicroscopy and green fluorescence protein (GFP) mRNA expression wastested by RT-PCR48h after transfection to determine the transfectionefficiency. Then the gene of hIL-12was amplified with PCR and the shuttleplasmid pAdTrack-IL-12was constructed. The shuttle plasmid wastransformed into Adeasier cell after linearization with Pme I digestion for homologous recombination in E. coli BJ5183. After digested with Pac I, therecombinant vector was transfected into AD293cells to packageadenovirus pAd-IL-12. The expression of IL-12was detected by RT-PCRand Western blot in human hepatocarcinoma cell Hep3B infected withpAd-IL-12virus. The proliferation of Hep3B was evaluated by MTT, andthe expression of TGF-β was detected with RT-PCR. Monocytes infectedby adenovirus carrying IL-12were indirectly co-cultured with Hep3B. Andthen detect the expression of CD206by flow cytometry to reflect thetumor-associated macrophages formation.Results: For K562and human monocytes, better transfectionefficiency was obtained by Ad5F35-GFP. All of the methods did nottransfect3T3-L1effectively. Hep A1-6could be transfected effectivelywith Ad5-GFP and Lipofectamine2000. The recombinant adenovirusplasmid containing IL-12was successfully constructed. Recombinantadenovirus pAd-IL-12were successfully constructed which could infectHep3B cell efficiently. In Hep3B infected with pAd-IL-12, highexpressions of IL-12mRNA and protein were confirmed by PCR andWestern blot, and both of the proliferation and TGF-βexpression ofHep3B were significantly reduced (both P<0.05) compared with controlgroup. The expression of CD206of the monocytes infected by adenoviruspAd-IL-12is lower than the control group, indicating that the formation ofTAM was reduced. Conclusions: Ad5F35-GFP could effectively transfect human K562and monocyte, which provides support for utilization of Ad5F35-GFP inrelative research and gene therapy involving human hematopoietic cellsand monocytes. The results demonstrate that recombinant adenoviruscontaining IL-12are successfully constructed. IL-12could suppress Hep3Bproliferation with the decline of TGF-βgene. Furthermore, IL-12couldreduce TAM formation in the tumor environment, which provides afoundation for further study of IL-12in tumor treatment.