摘要
背景与目的:B细胞淋巴瘤的独特型(idiotype,Id)免疫球蛋白可作为肿瘤特异性抗原诱发免疫反应抑制肿瘤的发展。本实验拟探讨小鼠淋巴瘤细胞株A20中Id-CTL表位的存在,了解细胞穿透肽(cell-penetratingpeptide,CPP)负载的Id(CPP-Id)进入树突细胞(dendriticcell,DC)的量,以及在细胞内的分布和进入细胞的时相,同时检测其对DC表面标志表达的影响。方法:用RT-PCR方法扩增A20细胞株的Id抗原CTL(cytotoxicitylymphocyte)表位的基因片段,并测序确定氨基酸的序列;通过流式细胞仪检测CPP-Id与Id进入小鼠DC的量的差异及抗原负载前后的DC表型的表达;共聚焦显微镜观察CPP-Id进入细胞的过程及时间;荧光显微镜观察CPP-Id在细胞内的分布。结果:RT-PCR扩增产物为660bp的片段,测序结果表明A20细胞株的Id抗原CTL表位的基因存在。共聚焦显微镜检测显示CPP-Id能在最初200s内快速进入DC,单纯Id进入DC的量很少。10μmol/L的CPP-Id负载的DC细胞平均荧光强度(MFI)高于单独使用Id组(4.35±0.48vs.1.14±0.33,P<0.005);两者负载的DC表面标记CD80、CD86、CD54及MHC-Ⅰ、MHC-Ⅱ类分子表达均比未成熟DC明显增加。结论:CPP-Id比Id进入小鼠DC的量明显增加,CPP肽可以提高Id抗原进入细胞的效率;CPP-Id体外冲击DC后,可以使DC表面MHC-Ⅰ、MHC-Ⅱ类分子,协同刺激分子CD80、CD86以及粘附分子CD54表达显著增高,有利于活化CTL。
BACKGROUND ＆ OBJECTIVE： Idiotypic immunoglobin （Id） derived from B-cell lymphoma, as a tumor-specific antigen, can suppress tumor development by inducing immune response. This study was to confirm the presence of Id epitope on cytotoxic T lymphocytes （Id-CTL） in mouse lymphoma cell line A20, detect the quantity and distribution of cell-penetrating peptide-loaded Id （CPP-Id） in dendritic cells （DCs） at different time points, and explore its impact on the expression of surface molecules on DCs. METHODS： Id-CTL epitope in A20 cells was amplified by reverse transcription-polymerase chain reaction （RT-PCR） and sequenced. The quantity of CPP-Id and Id alone in DCs and the expression of DC surface molecules were detected by flow cytometry. The process of CPP-Id entering DCs was observed under confocal microscope, and its distribution was observed under fluorescent microscope. RESULTS： A 660 bp-length fragment was amplified from A20 cells by RT-PCR, and identified as Id-CTL epitope by sequencing. CPP-Id entered DCs quickly during the initial 200 s, but few Id entered DCs. The mean fluorescent intensity （MFI） in DCs was significantly higher in CPP-Id group than in Id group （4.35±0.48 vs. 1.14±0.33, P〈0.005）. The expression of surface molecules CD80, CD86, CD54 and MHC-Ⅰ, MHC-Ⅱ were higher on the DCs loaded by either CPP-Id or Id alone than on immature DCs. CONCLUSIONS： CPP can enhance the quantity of Id entering DCs. CPP-Id can up-regulate the expression of MHC Ⅰ, MHC-Ⅱ and CD80, CD86, and CD54 on DCs, and may help to activate CTLs.
出处
《癌症》
SCIE
CAS
CSCD
北大核心
2005年第12期1484-1488,共5页
Chinese Journal of Cancer
