Investigation Of Anti-tumor Effects Of Lentivirus-mediated Tumor Vaccine By Constructing The Modified Human GM-CSF Gene Lentiviral Vector

Objective: Constructing the lentiviral vector encoding human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene, to approach whether the killing activity of cytotoxic T lymphocyte (CTL) was activated by the DC vaccine which was transfected with lenti-hGM-CSF was enhanced.Methods: 1) The DNA fragment of hGM-CSF was cloned from the plasmid pORFhGM-CSF with PCR and was inserted into the delivery vector L166 at the restriction nuclease sites BamH I and Xho I directionally to construct the recombinant vector L166- hGM-CSF. 2) The recombinant vector L166- hGM-CSF, the packing vector L205 and VSVG L311 were cotransfected into lentivirus packaging cell line 293T using the CaCl₂ method. After 72 hours, the lentivirus-producing supernatant was gained. We named it lenti-hGM-CSF. We gained the lenti-GFPs which encoding green fluorescence protein by the same process. 3) Hela cells were transfected with lenti-GFP in different concentration and the titres were determined. 4) The level of hGM-CSF secreted by the Hela cells transfected with lenti-hGM-CSF was tested using ELISA method. 5) Cord blood was collected to isolate monocytes which were cultured in medium supplemented rhGM-CSF, rhIL-4 and α-TNF. Suspending cells were collected to analyze their phenotypes by flow cytometer. The ability of dendritic cells stimulating proliferation of allogeneic T lymphocytes was detected by MTT. 6) Tumor lysate was obtained after rapid freeze/thaw exposures. Dendritic cells charged tumor lysate and dendritic cells charged tumor lysate which were transfected with lenti-hGM-CSF were cocultured with T lymphocytes. The killing activity of cytotoxic T lymphocytes (CTLs) activated by these DC vaccines were assayed by MTT method.Results: 1) We had inserted the hGM-CSF gene into the delivery vector L166 at the restriction nuclease sites BamH I and Xho I to construct the recombinant vector L166- hGM-CSF; 2)The titration of lenti-GFP was 3.2×10⁶IU/ml; 3) The human GM-CSF was presented highly in the supernatant of Hela cells transfected with lenti- hGM-CSF, which was kept for more than two months. 4) There were a large number of suspending cells in medium after three to five days' cultures. These cells were irregular and had plenty of thorns, which were typical form of dendritic cells. 5) The ability of stimulate mixed lymphocyte reaction and anti-tumor effects of dendritic cells charged tumor lysate were obviously enhanced after transfected with lenti-hGM-CSF.Conclusions: 1) The lentivirus vector encoding hGM-CSF gene was constructed. The gene-phored was expressed long-time and lasting in host cells, then expressing effects was very high and rapid; 2) Isolated DC successfully from cord blood and confirmed by phase contrast microsope and flow cytometer; 3) Transferred the human GM-CSF to dendritic cells using the lentiviral vector to produce GM-CSF DC vaccine. Incubation with tumor lysate could increase the antigen presentation of APC, accordingly enhanced the anti-tumor effects in vitro.