Study On Anti-tumor Effect Of Exosome Derived From AFP Gene-modified Dendritic Cells

Objective：Hepatocellular carcinoma（HCC）is a common malignancy with highrecurrence rate and poor prognosis in China.Due to lacking of effective methods forearly diagnosis,the treatment rates is very low.Now biotherapy is a promising strategyfor HCC treatment and attract intense attention in recent year.dendritic cells(dendritic cell, DC) vaccine is one of promising biotherapys and the reseach hasachievesd considerable progress, however, the outcomes of clinical application areless then expectation. biotherapy based on DC vaccine still face many deficiencies,so many scholars try to improve the situation,include of finding optimal pretreatmentof antigen.Currently,the commonly methods of antigen loading mothed includesynthetic peptide antigens,tumor lysates,viral vector which carry antigen gene, etc,gene modified DC to some extent can solve the problem of antigen source.however,the security of virus is not guaranteed in vivo. DC secreted exosomes are nanomericvesicles harboring functional proteins capable of promoting T cell immune responsesand tumor rejection,which has achieved good results in lung cancer andmelanoma.However, research of treatment of HCC is still in the exploratory stage. Inthis study, we successfully construted lentiviral carrying AFP gene,then modifyDC,isolate and purify the exosome from the DC culture supernatant, try to prepareDC vaccines and exosome vaccine, We also compared the efficiency of DC andDC-derived exosome in stimulation of AFP-specific CTL responses and antitumorimmunity. To provide the basis for the development of new subcellular vaccine andaviod lentiviral directly apply in vivo, to develop more promising tumor vaccine ofclinical application.methods:1. Applied lentivirus vectors systems and packaging cell Lenti-X293T,constructed lentiviral particles which can stablely express the target genes AFP. 2. Isolated monocytes from peripheral blood of healthy donor,.The monoeytes wereinduced with rhGM-CSF,rhIL-4,LPS. Cell morphology were observed under lightmicroscope and cell phenotypes were identified by FACS.3. loaded inmature DC respectively with synthesis AFP peptide, tumor lysates ofHepG2cells and lentiviral vector carrying AFP gene, To prepare DC vaccine.4．Exosome vaccine were isolated by ultrafiltrate the culture supernatant of matureDC followed by ultracentrifugation on a30%sucrose/deuterium oxide cushion,exosome was observed by transmission electron microscopy, function associatedmolecules analysis by western blot.5. The T lymphocyte proliferation effciency stimulated by DC vaccines and exosomevaccine was detected by WST-1, then assayed specific cytotoxic response induced byDC and exosome by LDH, detected the amount of INF-γ which secreted fromactivated T lymphocytes by ELLSA.6． Using exosome-AFP loaded DC,detected the anti-tumor immune activity byWST-1,LDH,ELISA.Results:1.Successfully constructed lentiviral vector PRV3-AFP, infected dendriticcells with efficiently secreted AFP proteins.2.Mature DC induced from peripheral blood appears a typical dendritic structure, theexpression of HLA-DR, CD80, CD83, CD86, CD1a molecules significantly higherthan immature DC.3.Apply synthetic AFP peptides, tumor lysates of HepG2cells,AFP lentiviralparticles load DC, to prepare DC vaccines.4.DC cell-derived exosomes (EXOs) are50to100nm diameter vesicles containingantigen-presenting molecules:HLA-DR、ICAM-1and CD86molecules.5.Lentivirus-infected DC stimulated T lymphocyte proliferation is significantly higherthan DC which treated by AFP peptide or tumor lysates (P <0.05), the cytotoxicityof HepG2cells which had stimulated by Lentivirus-infected DC was significantlyhigher than the other two groups (P <0.05). however,the amount of IFN-γ secreted byT lymphocytes appears no significant difference between the three groups.(P>0.05). 6. DC pretreated with exosome can induce more stronger antigen-specific Tlymphocytes-base immune responseConclusion:1. Successfully constructed AFP lentiviral vector, DC loaded byrecombinant lentiviral vectors can induce more effective anti-tumor immunity thenAFP peptides and tumor lysates,which is a more efficient way to prepare DC vaccine.2．Exosome with the ability of antigen presenting can induce proliferation anddifferentiation into cytotoxic T lymphocytes against HCC in vitro. DC loaded byexosome can induce more strong anti-tumor immune response.