Targeted Gene Modified Dendritic Cell Vaccine Therapy Of Transplanted Liver Cancer In Experimental Studies

OBJECTIVE To establish a method to generate and amplify dendritic cells(DC)from mouse bone marrow using the cytokine in vitro and identify their morphology and immunological phenotype.METHODS The dendritic cells(DC)were gotten after inducing the mouse bone marrow cells with granulocyte-macrophage colony-stimulating factor(GM-CSF,50ng/ml)and interleukin-4(IL-4,2ng/ml)at the sixth day,which were loosely adherent cells. The DCs were divided into two groups,one of them were analysised with Flow cytometry(FACS).The others were stimulated with tumor necrosis factor alpha (TNF-a,50ng/ml)of the culture and analysised with Flow cytometry 48 hours later,then the morphology of DCs was observed by light microscopy and electron microscopy.RESULTS The clusters of cells were gotten after inducing the mouse marrow mononuclear cells using rmGM-CSF and rmlL-4 for 24 hours in vitro,which increased significantly 3 days later.The suspension cells were collected at the seventh day.The cells were branching-like under inverted light microscope and electron microscopy,which was typical morphology of DC.The express of CD11c,MHCâ…¡,CD80 and CD86 of the cells induced with rmGM-CSF and rmlL-4 were detected by fluorescence activation cell sorting.The expressing rate of them of DCs were elevated after stimulating with rmTNF-a 48 hours.The expressing rate of them were[(81.60±10.02)%],[(78.70±11.35)%],[(65.30±5.73)%]and[(79.65±8.66)%]separately, which demonstrated that the DCs were maturate gradually after stimulating with rmTNF-a.CONCLUSION A large number of DCs can be gotten by inducing the mouse marrow mononuclear cells with rmGM-CSF and rmlL-4 in vitro,which were stimulated to mature by rmTNF-a. OBJECTIVE To construct a murine alpha-fetoprotein gene-trasfected dendritic cells vaccine(pAdBM5-mAFP-DC).METHODS The cell 293,a human embryonic kidney cell line as a packaging cell was used for culture of recombinant adenovirus vectors containing mAFP gene.The recombinant adenovirus was reproduced and purified.Virus titer was detected by TCID₅₀ method.DCs were differentiated from C57BL/6J murine bone marrow progenitor cells and induced and augmented by recombinant murine IL-4(rmIL-4)and recombinant routine GM-CSF(rmGM-CSF).DCs were transfected by recombinant adenovirus engineered with murine alpha-fetoprotein gent.MHCâ… ,MHCâ…¡,CD18a(LFA),CD54(ICAM),CD80(B7.1) and CD86(BT.2)molecule of the DCs were detected by FACS analysis before and after gene transfection.RESULTS The purified recombinant adenovirus pAdBM5-mAFP was obtained with total volume of 50μl and titer of 0.88×10⁸ PFU/ml.The DCs were transfected by recombinant adenovirus pAdBM5-mAFP. The expressing rate of MHCâ… ,MHCâ…¡,CD18a(LFA),CD54(ICAM),CDS0(B7.1) and CD86(B7.2)molecule of DCs were elevated significantly after transfection of mAFP gene,which were 69.3%,41.0%,42.1%,63.2%,39.4%and 38.6% separately(p＜0.05).CONCLUSION Culturing and reproduction of cell 293 can reproduce recombined adenovirus pAdBM5-mAFP which can be used to establish the tumour vaccine of pAdBM5-mAFP-DC after transfection of the DCs.The volume of pAdBM5-mAFP can meet the needs of gene transfection in vivo at laboratory study.DC vaccine engineered with mAFP had been constructed successfully,and it had a high level expression of MHCâ… ,MHCâ…¡, CD18a,CD54,CD80 and CD86 molecule after transduction compared with before transduction. OBJECTIVE To investigate the effect of pAdBM5-mAFP-DC tumour vaccine on the inhibition of implanted liver cancer in C57BL/6J mice. METHODS 40 C57BL/6J mice were randomly divided into group A,B,C,D and E(8 mice each group).Group A was inoculated mAFP gene-transfected DC(pAdBM5-mAFP-DC);group B was inoculated with simple pAdBM5-mAFP plasmid DNA;group C was inoculated with simple pcDNA3.1(+)-mAFP plasmid DNA.Group D was inoculated with simple DC,and group E is simply injected with PBS.Each mouse of group A and D was immunized with 5×10⁵ cells(0.1ml per one time)administered s.c.in the rignt axillary.Each mouse of group B and C was immunized with 10.0μg plasmid DNA(0.1ml per one time) administered s.c.in the rignt axillary.Each mouse of group E was injected with 0.1ml PBS.Each mouse was accepted vaccination vaccine one time per 1w,and immuned continually 4 times.All mice were injected with Hepal-6 cells at the third week after initial immunity.The growth of implanted tumor in mice was observed.Serum were collected from tumor-bearing mice and measured for TNF-a and IFN-γby ELISA at the 14^thdays after being implanted tumor cells.At the same time all mice were killed and tumor tissues were assessed by histopathology examination.RESULTS The mAFP transgenic DC tumor vaccine could induce powerful immunoprotection,immunized mice could effectively resist the attack of Hepal-6 hepatocarcinoma cell,the tumor's growth was delayed(three mice of group A never generate tumor).The tumor weight of group A,group B,group C,group D and group E were (0.05475±0.029148)g,(0.25425±0.092698)g,(0.14025±0.081074)g,(0.10400±0.027092)g and(0.29300±0.134784)g.The tumor weight of group A were the lowest among all groups.It has statistics significance that the tumor weight of group A compared with group B,group C and group E(p＜0.05).The tumor suppression rate of group A was 81.31%which was higher than other groups signifcantly.The levels of TNF-a and IFN-γin the A and D groups increased signifcantly which were(180.25±30.25,155.45±24.37)pg/ml and (105.32±12.57,90.33±9.70)pg/ml separately.And it has statistics significance that group A and D compared with each other(p＜0.05).The histopathology inspection indicated that inflammatory cells infiltration in tumor tissues were found in all groups,especially for group A,which is chiefly infiltrated by lympholeukocytes.Morevere,duc(?)the tumor tissues grown too fast without enough blood supply,the ischemic necrosis occured in most of the gruops except group A.CONCLUSION The mAFP transgenic DC tumor vaccine can inhibit the growth of tumor by inducing the T cells which can infiltrate and kill tumor cells through sitmulating the anti-tumor immune response,which can elevate the level of TNF-a and IFN-γin serum of the tumor-bearing mice.