【摘要】：目的：了解PML-RARα多肽和蛋白誘導(dǎo)臍血T細(xì)胞的T細(xì)胞受體(TCR)Vβ譜系表達和克隆性情況，，并分析其誘導(dǎo)的T細(xì)胞的特異性殺傷效應(yīng)。并構(gòu)建具有抗急性早幼粒細(xì)胞白血病(APL)作用的PML-RARα基因疫苗。 方法：分別以NB4細(xì)胞和不同濃度(16.7μg／ml，33.3μg／ml或50μg／ml)的PML-RARα多肽聯(lián)合IL-2、抗CD3單抗以及抗CD28單抗誘導(dǎo)臍血T細(xì)胞增殖，利用RT-PCR-基因掃描技術(shù)分析誘導(dǎo)一定時間(第5、10天或第3、6、9、12天)的臍血TCR Vβ亞家族的利用和克隆性增殖的特點，同時利用LDH法檢測其細(xì)胞殺傷性。用RT-PCR方法擴增PML-RARα基因序列，分別克隆到真核表達載體pIRES和分泌載體pSecTag中。將經(jīng)測序鑒定正確的重組質(zhì)粒轉(zhuǎn)染K562細(xì)胞，RT-PCR方法檢測重組質(zhì)粒在真核細(xì)胞中的轉(zhuǎn)錄情況，點雜交和SDS-PAGE凝膠電泳檢測蛋白的表達情況。 結(jié)果：限制性表達和克隆性增殖的TCR Vβ亞家族T細(xì)胞均可見于PML-RARα多肽和NB4細(xì)胞誘導(dǎo)后的臍血T細(xì)胞，經(jīng)LDH法檢測其細(xì)胞殺傷活性，顯示此T細(xì)胞具有較強的細(xì)胞毒作用。成功構(gòu)建了PML-RARα／pIRES和PML-RARα／pSecTag重組質(zhì)粒，經(jīng)測序鑒定二者序列均正確，將其轉(zhuǎn)染K562細(xì)胞后經(jīng)RT-PCR電泳鑒定表明在轉(zhuǎn)錄水平能夠表達，經(jīng)點雜交檢測顯示重組質(zhì)粒在真核細(xì)胞中可表達PML-RARα蛋白，經(jīng)SDS-PAGE電泳檢測在預(yù)期的位置亦可見一目的條帶，大小約為17KD。 結(jié)論：PML-RARα多肽和NB4細(xì)胞可在體外誘導(dǎo)臍血T細(xì)胞呈克隆性增殖，此優(yōu)勢增殖的克隆性T細(xì)胞具有特異性細(xì)胞毒作用。成功構(gòu)建PML-RARα融合基因疫苗，體外真核細(xì)胞中能表達PML-RARα蛋白。
[Abstract]:Aim: to investigate the expression and cloning of T cell receptor (TCR) V 尾 in cord blood T cells induced by PML-RAR 偽 peptide and protein, and to analyze the specific cytotoxicity of T cells induced by PML-RAR 偽 peptide and protein. PML-RAR 偽 gene vaccine with anti-(APL) effect in acute promyelocytic leukemia was constructed. Methods: cord blood T cell proliferation was induced by NB4 cells and different concentrations (16.7 渭 g / ml 33.3 渭 g/ml or 50 渭 g/ml) of PML-RAR 偽 peptide combined with IL-2, anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody, respectively. RT-PCR- gene scanning technique was used to analyze the utilization and clonal proliferation of TCR V 尾 subfamily in umbilical cord blood induced for a certain period of time (5th day, 10th day or 3rd day, 912 day). Meanwhile, LDH assay was used to detect the cytotoxicity of human umbilical cord blood TCR V 尾 subfamily. PML-RAR 偽 gene was amplified by RT-PCR and cloned into eukaryotic expression vector pIRES and secretory vector pSecTag, respectively. The transcription of recombinant plasmid in eukaryotic cells was detected by RT-PCR, and the expression of protein was detected by dot blot and SDS-PAGE gel electrophoresis. Results: the restricted expression and clonal proliferation of TCR V 尾 subfamily T cells could be found in PML-RAR 偽 peptide and NB4 cells induced T cells in umbilical cord blood. The cytotoxicity of these T cells was detected by LDH assay. The recombinant plasmids PML-RAR 偽 / Pires and PML-RAR 偽 / pSecTag were successfully constructed. The sequence of the two recombinant plasmids was confirmed by sequencing. After transfection into K562 cells, RT-PCR electrophoresis showed that they could be expressed at the transcriptional level. Dot blot analysis showed that the recombinant plasmid could express PML-RAR 偽 protein in eukaryotic cells, and a target band was also found by SDS-PAGE electrophoresis. The size of the recombinant plasmid was about 17kD. Conclusion the cloned T cells of cord blood can be induced to proliferate in vitro by NB4 cells and 1: PML-RAR 偽 polypeptide. The dominant clonal T cells have specific cytotoxic effects. PML-RAR 偽 fusion gene vaccine was successfully constructed and expressed PML-RAR 偽 protein in eukaryotic cells in vitro.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R392

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