NY-ESO-1致敏树突状细胞诱导的CTL杀伤视网膜母细胞瘤细胞株作用的研究

发布时间：2018-03-16 20:35

本文选题：视网膜母细胞瘤　切入点：树突状细胞　出处：《重庆医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：研究目的：本文通过肿瘤睾丸抗原NY-ESO-1致敏树突状细胞，诱导特异性细胞毒性T淋巴细胞对视网膜母细胞瘤细胞株HXO-RB44的杀伤作用效果。研究方法： 1.自行构建NY-ESO-1基因的重组质粒pDC316/NY-ESO-1，并鉴定其在真核细胞中的表达。 2.Ficoll法分离人血获得单个核细胞，通过rhGM-CSF和rhIL-4诱导培养外周血来源的DCs，重组质粒转染DCs，LPS诱导DCs成熟，成熟DCs刺激特异性CTL细胞增殖。CTL特异性杀伤HXO-RB44细胞株，通过MTT法检测CTL的增殖以及CTL对视网膜母细胞瘤细胞的杀伤作用。研究结果： 1.经过检测重组载体pDC316/NY-ESO-1的目的片段序列与原基因序列完全相同，酶切鉴定结果与预期一致。 2.在细胞株HXO-RB44中可以检测到NY-ESO-1基因的表达，Western-blot法和免疫荧光可检测到NY-ESO-1基因较为强烈的表达。 3.经rhGM-CSF和rhIL-4成功诱导出的外周血DCs，脂质体介导重组质粒pDC316/NY-ESO-1转染诱导成熟的DCs，通过流式细胞技术检测到转染后DCs表型分子分别是HLA-DR为42.1%,CD80为54.2%，CD83为39.7%，CD86为94.8%。转染后DCs刺激产生的CTL组的增殖能力强于未经过转染DCs刺激产生的CTL组。 4.通过将转染后DCs诱导的CTL，未转染DCs诱导的CTL和无DCs的淋巴细胞三组分别同细胞株HXO-RB44进行联合培养，检测到转染后DCs刺激产生的CTL对视网膜母细胞瘤细胞株的杀伤效果显著高于未经处理DCs刺激产生的CTL组和无DCs组，且效靶比为75:1(P0.05)时杀伤率最强。结论：本实验检测到NY-ESO-1基因在细胞株HXO-RB44中较为强烈的表达，NY-ESO-1在CT抗原中是最能引起体液和细胞免疫反应的抗原，并且也是最具免疫原性的肿瘤抗原之一，通过NY-ESO-1重组质粒构建，脂质体介导重组质粒转染DCs诱导的特异性CTL产生，而此种CTL对细胞株HXO-RB44具有特异性杀伤作用，，为RB免疫治疗提出了一条新的方向，然而此种方法处于探索性研究阶段，尚需进一步深入研究。
[Abstract]:Objective: to induce specific cytotoxic T lymphocytes (CTLs) to induce cytotoxic effects on retinoblastoma cell line (HXO-RB44) by sensitizing dendritic cells with tumor testicular antigen (NY-ESO-1). Research methods:. 1.Recombinant plasmid pDC316 / NY-ESO-1 of NY-ESO-1 gene was constructed and its expression in eukaryotic cells was identified. 2. Mononuclear cells were isolated from human blood by Ficoll method. DCS derived from peripheral blood were induced by rhGM-CSF and rhIL-4. The recombinant plasmid was transfected with DCs to induce DCs maturation, and mature DCs stimulated the proliferation of specific CTL cells. CTL-specific killing of HXO-RB44 cells was observed. The proliferation of CTL and the cytotoxicity of CTL to retinoblastoma cells were detected by MTT assay. Results of the study:. 1. The sequence of the target fragment of the recombinant vector pDC316/NY-ESO-1 was identical to that of the original gene, and the result of restriction endonuclease digestion was consistent with the expected result. 2. The expression of NY-ESO-1 gene could be detected by Western-blot and immunofluorescence in HXO-RB44. 3. RhIL-4 and rhGM-CSF were successfully induced in peripheral blood DCS and liposome-mediated recombinant plasmid pDC316/NY-ESO-1 was transfected into mature DCS. The phenotypic molecules of DCs after transfection were detected by flow cytometry to be HLA-DR = 42.1 and 54.2% respectively. CD83 was 39.7i and CD86 was 39.7%. After transfection, DCs stimulation was achieved. The proliferative ability of CTL group was stronger than that of CTL group without DCs stimulation. 4. The CTLs induced by DCs, CTL cells without DCs and lymphocytes without DCs were co-cultured with the cell line HXO-RB44, respectively. The killing effect of CTL stimulated by DCs on retinoblastoma cell line was significantly higher than that of CTL group and DCs free group induced by untreated DCs, and the killing rate was the strongest when the ratio of effect to target was 75: 1 (P0.05). Conclusion:. In this study, we found that NY-ESO-1 gene is strongly expressed in HXO-RB44 cell line. NY-ESO-1 is one of the most immunogenicity tumor antigens in CT antigens. NY-ESO-1 is one of the most immunogenicity tumor antigens, and is constructed by NY-ESO-1 recombinant plasmid. Liposome-mediated recombinant plasmid transfection of DCs induces specific CTL production, and this kind of CTL has a specific killing effect on cell line HXO-RB44, which provides a new direction for RB immunotherapy. However, this method is still in the exploratory stage. Further research is needed.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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