荷CEA-rV的DC增强CD3AK对CEA阳性肿瘤特异杀伤作用的研究  

Special killing effect of CD3AK On anti -CEA-positive tumor enhanced by DC loaded with CEA-rV

作　　者：祁岩超[1] 王新帅[1] 扬波[1] 卢敏莹[1] 潘东晓[1] 刘枫[1] 

机构地区：[1]广州医学院临床肿瘤研究中心暨广州市肿瘤医院,广东广州510095

出　　处：《世界肿瘤杂志》2005年第4期245-249,261,F0004,共7页Tumour Journal of the World

基　　金：广东省科技厅科研基金资助（2002C30305）

摘　　要：目的 观察荷CEA-rV的DC增强CD3AK细胞对CEA阳性肿瘤细胞的特异性杀伤功能。方法 用脐血制备脐血单个核细胞（UBMC），培养3h后，收集悬浮细胞制备CD3AK，重悬贴壁细胞制备DC。DC培养3d时加入CEA-rV，制备CEA-vV＋DC；在CD3AK培养8d时，加入CEA-rV＋DC混合培养，制备CEA-rV＋DC＋CD3AK。用MTT法测效应细胞UBMC，CD3AK，DC＋CD3AK，CEA-rV＋DC＋CD3AK，分别对CEA阳性靶细胞：LOVO，A549和CEA阴性靶细胞：肝癌细胞株BEL-7402，红白血病细胞株K652的杀伤活性。结果 ①用CEA兔抗人单克隆抗体对四种靶细胞株的鉴定结果表明，LOVO和A549确是CEA阳性细胞株，BEL．7402和K562确是CEA阴性细胞株。②培养10d的DC流式细胞仪检测结果示：高表达MHCI，Ⅱ类分子CD86（82．7％），CD80（51．1％），CD83（57．5％），CD40（69.4％），低表达CD123分子，光学和电子显微镜观察，DC细胞呈不规则形，有大量突起和伪足，成熟DC直径15-20um，胞浆线粒体，内质网丰富。证明为成熟DC。③CD3AK细胞和单纯UBMC细胞的流式细胞仪的免疫分子表型结果是，无论CD3，CD4，CD8和CD28，在CD3AK细胞组中的比率都是UBMC组的二倍以上。说明CD3AK组中成熟T淋巴细胞量明显高于UBMC细胞组。④三种效应细胞CEA-rV＋DC＋CD3AK，DC＋CD3AK和CD3AK对四种靶细胞的杀伤率均比单纯UBMC组明显提高（P〈0.01），而且CEA-rV＋DC＋CD，AK组〉DC＋CD3AK组〉CD3AK组〉UBMC组。说明细胞因子，DC和CEA-rV都有进一步提高UBMC广谱的杀伤肿瘤细胞活性的作用。⑤CEA-rV＋DC＋CD3AK和CD，AK相比较，对CEA阳性细胞LOVO和A549的杀伤活性提高的显著性（P〈0．01）明显优于对CEA阴性细胞K562和BEL-7402显著性（P〈0．05）。说明CEA-rV＋DC能显著提高CD3AK细胞对CEA阳性肿瘤的特异杀伤活性。CEA-rV＋DC＋CD3AK组和DC＋CD3AK组相比，对CEA阴性细胞株的杀伤活性无显著差异（P〉0．05），而对CEA阳�Objective To survey the special killing activity of CD3AK on anti-CEA-positive tumor enhanced by umbilical cord blood dendritic cell （DC） loaded with CEA recombinant vaccinia virus （CEA-rV）. Methods Freshly isolated umbilical blood mononuclear cells （UBMC） were cultivated for 3 hours. Suspension cells and attached cells were used to induce CD3AK cells and DC, separately. DC was loaded with CEA-rV on the 3^rd day to prepare CEA-rV＋DC. CD3AK cells were co-cultured with CEA-rV＋DC on the 8th day, to prepare CEA-rV＋DC＋CD3AK. The killing activity of each effector＇s cell, which included UBMC, CD3AK, DC＋CD3AK and CEA-rV＋DC＋CD3AK, was measured respectively by MTT reduction assay. There are CEA positive tumor cell and CEA negative tumor cell in the target cell groups. Lovo cell that is a colon carcinoma cell line and A549 cells that is a lung cancer cell line are CEA positive cell lines; Bel-7402 cells that is a hepatoma carcinoma cell line and K562 cells that is an erythroleukemia cell line are CEA negative cell lines. Results ①4 target cells were confirmed by CEA monoclonal antibody of rabbit anti-human. Lovo and A549 are really CEA positive cell lines, while Bel-7402 and K562 are CEA negative cell line. ②It was showed by flow-cytometry that the mature DC cultured at 10^th day expressed MHC Ⅰ, Ⅱ molecule such as CD86, CD80, CD83 and CD40 high, but CD 123 low. The express rate of CD86, CD80, CD83 and CD40 was 82.7%, 51.1%, 57.5% and 69.4% respectively. The appearances and intra-cellular structures of DC were observed through light and electron microscope. The diameter of mature DC was 15-20μm presented the irregular morphologic appearance, much prominences and pseudopodium. There were abundant mitochondria and endoplasmic reticulum in DC endochylema. ③The rate CD3, CD4, CD8 and CD28 of in CD3AK cells group was 2 folds higher than that in UBMC group by FACS. It is said that the number of the mature T lymphocyte in CD3AK cells group was much greater than that in UBMC group. ④The kill

关 键 词：CEA重组痘苗病毒 树突状细胞 CD3AK细胞 CEA阳性肿瘤细胞 

分 类 号：R730.21[医药卫生—肿瘤]