摘要
背景目前用细胞免疫的方法治疗视网膜母细胞瘤（RB）成为新的研究热点，肿瘤-睾丸抗原是最具免疫原性的人类肿瘤抗原之一，已用于全身一些肿瘤的免疫治疗，但其对RB治疗作用的研究报道较少。目的探讨肿瘤-睾丸抗原的NewYork—esophageal-1（NY—ESO-1）致敏树突状细胞（DCs）诱导特异性细胞毒性T淋巴细胞（CTL）对RB细胞株HXO—RB44的杀伤作用。方法用聚合酶链反应（PCR）技术对NY—ESO-1质粒的目的基因片段进行扩增，使用SalⅠ限制酶和EcoRI限制酶进行双酶切，切胶回收DNA片段。将其连接入pDC316质粒，构建pDC316／NY—ESO．1真核表达载体并再进行酶切鉴定。免疫荧光及Western blot法检测NY—ESO-1蛋白在HXO．RB44细胞株中的表达。Ficoll法分离血获得单个核细胞，通过RPMI1640重悬，调整细胞密度为1×10。个／ml，通过重组人集落刺激因子（rhGM—CSF）和重组人白细胞介素4（rhlL-4）诱导培养外周血来源的DCs，通过重组质粒转染DCs，以脂多糖诱导DCs成熟，DCs与淋巴细胞数量比为1：25的比例混合培养HXO．RB44细胞，通过MTT法检测CTL的增生情况。在培养液中同时加入CTL细胞，MTT法检测HXO—RB44细胞的活力。结果重组质粒pDC316／NY—ESO-1的目的片段序列与NY-ESO-J基因序列完全相同，酶切鉴定结果与预期一致。Western blot法以及免疫荧光检测结果显示，NY—ESO．1在细胞株HXO．RB44中呈强阳性表达。经rhGM．CSF和rhlL-4成功诱导出的外周血DCs，重组质粒转染后其表型分子分别是HLA—DR为42．1％，CD80为54．2％，CD83为39．7％，CD86为94．8％。MTT法检测表明，pDC316／NY—ESO-1质粒转染的DCs诱导的CTL组的增生能力强于未转染组，致敏的DCs与淋巴细胞比例为1：100时诱导CTL效果最为显著，与未转染组比较差异有统计学意义（P〈0．05），CTL对肿瘤细胞的杀伤作用明显强于未转染组和无DCs组，且效靶比为75：1时杀伤率最强，差异有统计学意义（P〈0．05）。结论NY—ESO-1质粒转染DCs诱导的CTL对RB细胞具有特异性杀伤作用，为RB免疫治疗提供了一种新的方法。
Background Cell immunologic therapy for retinoblastoma （RB）is becoming a hot research topic. Cancer-testis antigen is a human immunogenic protein and is used to treat some tumors. However,its effect on RB has not been investigated. Objective The present study was to discuss the antigen specific anti-tumor effect of cytotoxic T lymphocytes （CTL）induced by the cancer-testis antigen, NY-ESO-l-sensitized dendritic cells （DCs）, on human RB. Methods PCR was performed to amplify target gene fragments from the NY-ESO-1 plasmids,and then the target gene fragments were digested with the restriction enzymes SalI and EcoRI. Harvested fragments were inserted into the pDC316 plasmid to construct the recombinant plasmid pDC316/NY-ESO-1. The expression of NY-ESO-1 protein in human RB cells strain, HXO-RB44,was detected by immunofluoreseence and Western blot. Monoeytes wereisolated from 60 ml of peripheral blood from a healthy donor using Ficoll density-gradient centrifugation with a cell density of 1 x 107/ml. DCs isolated from blood were stimulated with recombinant human granulocyte-macrophage colony stimulating factor （rhGM-CSF） and recombinant human interleukin-4 （ rhIL-4 ）. The recombinant plasmid pDC316/NY-ESO-1 was transfected into DCs and the DCs were co-cultured with T lymphocytes. The resultant CTL were used as effector ceils. The growth of the CTL was detected by MTT assay. The CTL were then added into the growth medium used for culturing HXO-RB44 cells and the vitality of the HXO-RB44 cells was assayed by MTT assay. Results The sequence of the cloned DNA fragment of the recombinant plasmid pDC316/NY-ESO-lwas conforms with the sequence of the NY-ESO-1 gene. The expression of the NY-ESO-1 protein in HXO-RB44 cells was tested by immunofluorescence and Western blot. DCs were successfully induced with rhIL-4 and rhGM-CSF from PBMC. The recombinant expression plasmid pDC316/NY-ESO-1 was successfully transferred into DCs. These DCs had high expression of surface molecules such as HLA-DR （42. 1% ） , CD80 （54.2%） , CD83 （39.7%） and CD86 （94.8%）. The CTL that was induced by DCs-sensitized with NY-ESO-1 specifically killed HXO-RB44 cells. CTL induced by the sensitized DCs had a stronger cytotoxic effect against HXO-RB44 cells compared with un-sensitized DCs and CTL un-induced with DCs, as shown by MTT asssay（ P〈0.05 ）. The anti-tumor activity was highest when the ratio of effector to target was 75 ： 1 （ P〈0. 05 ）. Conclusions DCs transfected by the recombinant plasmid pDC316/ NY-ESO-1 can induce the proliferation of allogenic CTLs, which showed a specific anti-tunmr effect against HXORB44 cells. These results present a new type of immunotherapy for the treatment of RB.
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2012年第7期586-591,共6页
Chinese Journal Of Experimental Ophthalmology
基金
国家自然科学基金项目（30872826）
