| [Objective] a -Fetoprotein is a tumor-associated differentiation antigen that is frequently expressed at high level in the majority of hepatocellular carcinoma(HCC), and is to be used as a target molecule for immunotherapy against the tumor. We investigated the potentiality of DNA vaccination with human AFP-expressing plasmid to evoke or amplify cytotoxic T lymphocytes (CTLs) against AFP-expressing tumor cells in mice. To get a better understanding the mechanism of DNA initiating specific CTL immune response, we also observed that local infiltration in injection site at different time after DNA immunization. The character of infiltration cells were further studied with anti-CD205, anti-CD4, anti-CD8 by immunohistological stain. Finally, we investigated the capacity of the myoblast as facultative APC.[ Methods 3 To study DNA immune responses, hAFP-expressing recombinant vaccine were generated. Total RNA was isolated from fetal liver tissue. Full-length human AFP cDNA was obtained by RT-PCR amplification and recombinant into eukaryotic expression plasmid pcDNA3.1.The sequence of the recombinant plasmid phAFP was determined. The AFP expressions in CHO transfected with phAFP and in muscle after injection phAFP were investigated by immunohistological methods. The tibialis anterior muscle of mice were injected, and boosted biweekly for 4 weeks with combination of phAFP and phGM-CSF, phAFP alone, pcDNA3.1, and phGM-CSF or 0.01mMPBS, respectively. An HCC tumor model was established in BALB/cmice by injection AFP-expressing H22 cells. The studies were performed through two basic strategies: hAFP plasmid-based protective antitumoral immunity and therapeutic antitumoral immunity.In the first strategy, mice were injected into the right flank of mice with 1×105 H22 cells after two weeks immunization. Tumor incidence and volume were evaluated once a week using calipers. Spleen cells were harvested 2 weeks after the final immunization. Proliferation of T lymphocyte was detected in 3H-TdR. The specific lysises of CTL and NK were assayed against YAC-1 target cells or H22 target cells in a 51Cr-release assay at 1:50 E/T ratio respectively. Serum were collected from tumor-bearing mice and measured for TNF a, IFN γ by ELISA at 3 and 6 weeks initial-post immunization.We continually evaluated the therapeutic antitumoral immunity. After one week of injection H22 cells, the tumor-bearing mice were immunized as well as the protection vaccination. Tumor size, the activity of NK and AFP-specific CTLs, the proliferation of T lymphocyte and cytokine levels in DNA immunized mice were assayed as indicated above.We also observed the local infiltration in injection sites at 6 hour, 12hour, on day 1 to 6 by H&E stain after DNA immunization. Infiltration cells were further studied with the mice dendritic cells marker CD205, T lymphocyte marker CD4 and CD8 by immunohistological methods. In addition, we investigated the capacity of the myoblast cultured from mice muscle, acting as facultative APCs. Myoblasts were cultured and purified to >95%, The MHC class I and II expression on myoblast were detected by FACS before and after induction with different concentrations IFN γ .[Results] The restriction endonuclease mapping of recombinant plasmid showed 1.8kb fragments as well as full-length human AFP cDNA, and the sequence is consistent with it from GenBank. The phAFP was successfully expressed in CHO and in muscle tissues. We established a reliable and reproducible HCC tumor model. Then we tested the protective and therapeutic efficacy of the DNA immunization against AFP through based two strategies. We first assessed the protective antitumoral activity. It was an important finding that all mice specifically immunized against hAFP showed a delayed tumor growth, significant activity of CTL and NK, and strong proliferation of T lymphocyte. The levels of IFN Y and TNF a increased significant high compared to mice vaccinated with control plasmid at 6 weeks after immunization, although no differences was observed in each gr...
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