| Objective:To construct recombinant plasmid IP-10/pcDNA3.1 by inserted IFN-y inducible protein-10 (IP-10) DNA fragments into pcDNA3.1(+) vector. To establish the methods of isolation, purification and propagation of mouse bone marrow-derived dendritic cell (DC), investigate the changes of morphological structure and bio-immunological characteristics of DC during its development, differentiation and maturation, study the related factors involved in proliferation and polarization of DC. To construct the DC vaccine pulsed with whole tumor cell lysate and modified with IP-10 gene. To detect and analyze the specific anti-tumor activity, the immunotherapeutic function and immune protection of DC vaccine both in vitro and in vivo,Methods:The cDNA fragment encoding the murine IP-10 was obtained from mRNA of murine T cells by using Reversal transcript-polymerase chain reaction (RT-PCR) and contained a signal peptide derived from the human monocyte chemoattractant protein 1, then it was inserted into the eukaryotic expression vector pcDNA3.1(+). The mouse bone marrow progenitors were isolated and cultured with cytokines of GM-CSF plus IL-4, and a large amount of BM-DC were harvested after special selection and culture for 7 days. The bio-immunological features and functions of DC in different stages were studied by using methods of light- and electro-microscopy, HRP phagocytosis assay, mixed leucocyte reaction (MLR), flow cytometry (FCM), and so on. Mouse bone marrow DC were pulsed with cell lysate from RM-1 prostate cancer cell line and then transfected with the recombinant plasmid IP-10/pcDNA3.1 by DOTAP liposome. The expression of IP-10 was assayed by Western blot and chemotaxis assay. The ability of stimulating syngeneic T cells and the effect of inducing specific kill activity of DC vaccine were detected. The immunotherapeutic effect of DC vaccine on mice with prostate cancer was assessed. The immunological protection induced by DC vaccine was studied by the challenge of tumor cell injection followed the inoculation.Results:1. By partial nucleotide sequencing, the cDNA was consistent with the reported mIP-10 cDNA in the GeneBank, and then was inserted into the eukaryotic expression vector pcDNA3.1(+) to construct recombinant plasmid IP-10/pcDNA3.1.2. About 5~10X106 DC were harvested from bone marrow progenitors per mouse followed culturing the cells with GM-CSF plus IL-4 for 7 days. In early stage, DC possessed short processes and fewer organelles, but a large numbers of vacuoles in the cytoplasm. They expressed very low level of B7-1, B7-2, MHC class-I and class-II molecules and failed to stimulate allogeneic T cell proliferation, but could pino-phagocytose vigorously HRP granules. After 7 days culture, they exhibited morphologically the distinct dendritic shape with long processes, increased organelle and decreased fraction of vacuoles. Simultaneously, they expressed higher level of surface MHC and B7 molecules and stronger activity of stimulating T cell proliferation.3. The IP-10 plasmid vector was successfully transfected into DC and the DC tranfected with IP-10 gene were capable of synthesizing and secreting IP-10 chemokine, which could increase the preferential chemotaxis of DC to T cells. The DC vaccine modified with tumor cell lysate and IP-10 gene (IP-10/Lysate-DC) was a more potent stimulator in MLR. The mice immunized with DC pulsed with RM-1 cell lysate (Lysate-DC) exhibited a specific CTL response, but the highest CTL activity against RM-1 cells was induced by immunization with IP-10/Lysate-DC (p<0.01).4. In the mice model with pre-established subcutaneous RM-1 prostate cancer, vaccination with Lysate-DC could inhibit tumor growth, but the immunization of IP-10/Lysate-DC inhibited the tumor growth most significantly when compared with Lysate-DC (p<0.01). The survival time of the mice treated with IP-10/Lysate-DC was also greatly extended (p<0.01).5. Histological examination showed that the most obvious tumor nec...
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