Investigation Of Anti-tumor Effects Of Lentivirus-mediated And HTERT Gene Modified DC Vaccine

Objective:Constructing the lentiviral vector encoding human Telomerase Reverse Transcriptase(hTERT)gene,to investigate the killing activity of cytotoxic T lymphocyte(CTL)activated by the DC vaccine which was infected with Lenti-hTERT.Methods:1)Got the DNA fragment of hTERT from mRNA of HepG2 cell line by RT-PCR,then inserted it into delivery vector L166 to construct the recombinant vector L166-hTERT.2)The recombinant vector L166- hTERT,packing vector L205 and VSV-G vector L311 cotransfected lentivirus packaging cell line 293T using the CaCl₂ method.72 hours later,the lentivirus-producing supernatant was gained.We named it Lenti-hTERT.We also gained the Lenti-GFP which encoding green fluorescence protein by the same process.3)Infected 293T cells with Lenti-GFP in different concentration then the titres and infecting efficiency were determined. Detected the expression of hTERT gene in 293T cells infected with Lenti-hTERT using immunocytochemistry stain.4)Got isolate monocytes from cord blood and cultured in medium supplemented rhGM-CSF,rhIL-4 and rhTNF-α.Collected suspending cells to analyze their phenotypes by flow cytometry.5)Infected dendritic cells with Lenti-hTERT to construct DC vaccine.The ability of stimulating proliferation of allogeneic T lymphocytes and the killing activity of CTL activated by these DCs were detected with MTT method.Results:1)DNA sequencing showed that we had inserted the hTERT gene into the delivery vector L166.2)293T cells producing virus particles became bigger and swell,also the adhesive ability decreased.A lot of green fluorescence was observed in cells producing Lenti-GFP.3)The titration of Lenti-GFP was 5.88×10⁶IU/mL and the infecting efficiency was about 90%.hTERT gene could express in 293T cells infected by Lenti-hTERT and which could keep for more than two months.4)Isolate monocytes collected from cord blood became irregular and had plenty of thorns after three to five days' culture,which was a typical form of dendritic cells,and flow cytometry also showed dendritic cells' phenotypes.5)The DC vaccine modified by hTERT gene demonstrated fine ability of stimulating mixed lymphocyte reaction and anti-tumor effects.Conclusions:1)Constructed the lentivirus vector encoding hTERT gene successfully,and the gene-phored could express constantly at a high level in host cells. 2)Isolated DC from cord blood and confirmed by phase contrast microscope and flow cytometry.3)Transferred the hTERT gene to dendritic cells using the lentiviral vector and produced DC vaccine,which could increase the antigen presentation and stimulate the proliferation of allogeneic T lymphocytes,accordingly enhanced the anti-tumor effects in vitro.