| Objective: The first aim of this study was to investigate the expression and clonality of T cell receptor (TCR) Vβ repertoire in cord blood T cells induced by PML-RARα peptide and NB4 cells in vitro, and farther to analyze the specific cytotoxicity for APL in the induced T cells. The second aim was to develop a reconbinant PML-RARα gene vactor which was expected to be used as a DNA vaccine to display an anti-APL effect.Methods: Cord blood mononuclear cells were amplified by IL-2, anti-CD3 and anti-CD28 antibody with different concentration (16.7μg /ml, 33.3μg /ml or 50μg /ml respectively) of synthetic PML-RARα peptide or NB4 cells. The induced T cells were collected at different time points after culture (3, 6, 9 or 12 days or 5,10 days). The expression and clonality of TCR Vβ subfamilies within induced T cells were analyzed by using RT-PCR and genescan technique. And the leathl effection of the induced clonal T cells was detected by using LDH technique. The PML-RARα gene fragment were amplified using RT-PCR, and then were cloned into pIRES and pSecTag vectors respectively. The recombinant plasmids were analyzed by sequencing and were transfected into K562 cells. The expression of the PML-RARα mRNA and protein in transfected K562 cells were detected by RT-PCR dot blotting and SDS-PAGE gel electrophoresis respectively.Results: Restricted expression and cloanl expansion of TCR Vβ subfamily cord blood T cells could be identified after induction by PML-RARα peptide and NB4 cells, and the induced T cslls have strong specific cytotoxicity. The recombinant plasmids PML-RARα/pIRES and PML-RARα/pSecTag were developed and the sequences were confirmed by sequencing. The recombinant plasmids were transfected into K562 cells and the PML-RARα mRNA and protein could be identified in the transfected K.562 cells.Conclusion: The PML-RARα peptide and NB4 cells could induce the clonal expansion T cells from cord blood in vitro, which may have specific... |
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