黑色素瘤相关抗原-A3蛋白激活树突状细胞诱导CD8＋细胞毒性T淋巴细胞免疫治疗肝细胞癌  

Immunotherapy of hepatocellular carcinoma by melanoma - associated antigen - A3 activated dendritic cells-induced antigen -specific CD8＋ cytotoxic T lymphocytes

作　　者：王谦[1] 黄长山[1] 余伟[1] 丁月超[1] 马超[1] 黄涛[1] 

机构地区：[1]郑州大学附属肿瘤医院(河南省肿瘤医院)肝胆胰外科,450008

出　　处：《中华实验外科杂志》2017年第4期587-590,共4页Chinese Journal of Experimental Surgery

摘　　要：目的探讨黑色素瘤相关抗原-A3（MAGE-A3）蛋白激活树突状细胞（DC），然后诱导产生CD8＋细胞毒T淋巴细胞（CTL）进行免疫治疗肝细胞癌（简称肝癌）的效果。 方法利用MAGE-A3蛋白诱导DC成熟并检测DC表面标志物及白细胞介素（IL）-10、IL-12表达，然后利用成熟的MAGE-A3-DC诱导MAGE-A3特异性CD8＋CTL，并检测MAGE-A3-DC-CD8＋CTL对正常肝细胞L02和肝癌细胞HepG2的杀伤作用。建立BALB/c-nu/nu肝癌模型，检测MAGE-A3-DC-CD8＋CTL对小鼠肿瘤的抑制作用，并通过病理学切片观察肿瘤组织变化。 结果MAGE-A3蛋白刺激能够显著上调DC表面标志物DC80、DC83、DC86和人类白细胞抗原DR基因（HLA-DR）水平（97.45±2.58比48.33±1.68，92.81±2.36比52.92±1.90，94.00±3.01比53.97±2.05，92.16±1.87比34.13±1.32；t＝35.680，P＝0.013；t＝29.440，P＝0.021；t＝24.580，P＝0.012；t＝56.690，P＝0.009）；与磷酸盐缓冲液（PBS）刺激的DC比较，MAGE-A3蛋白能够显著促进DC释放IL-12[（338.44±18.15） pg/ml比（243.23±16.56） pg/ml；t＝8.670，P＝0.005]和显著抑制IL-10的释放[（207.21±10.89） pg/ml比（327.58±14.36） pg/ml；t＝14.930，P＝0.009]。MAGE-A3-DC-CD8＋CTL和DC-CD8＋CTL展示了相近的L02细胞杀伤效果[（9.10±1.40）%比（9.71±1.58）%；t＝0.650，P＝0.120]，与DC-CD8＋CTL比较，MAGE-A3-DC-CD8＋CTL展示了显著的HepG2细胞杀伤效果[（58.84±5.27）%比（9.63±1.61）%；t＝19.970，P＝0.008]。BALB/c-nu/nu肝癌小鼠经过MAGE-A3-DC-CD8＋CTL治疗后，肿瘤体积比显著低于PBS组和DC-CD8＋CTL组（5.43±1.22比21.81±2.01比22.85±2.40；t＝22.030，P＝0.010；t＝20.460，P＝0.012）。苏木素-伊红（HE）染色结果显示，经过MAGE-A3-DC-CD8＋CTL处理的肿瘤组织细胞核固缩，细胞间隙增大，出现空泡和水肿现象，而PBS组和DC-CD8＋CTL组肿瘤组织无病理学改变。结论MAGE-A3蛋白能够刺激DC细胞成熟并诱导产生MAGE-A3蛋白特�Objective To observe the effect of immunotherapy on hepatocellular carcinoma （HCC） by melanoma-associated antigen-A3 （MAGE-A3） activated dendritic cells （DC）-induced antigen-specific CD8＋ cytotoxic T lymphocytes （CTL） and offer help to HCC treatment. Methods After MAGE-A3 activated DC biomarkers, interleukin （IL）-12, and IL-10 in DC were tested. MAGE-A3-DC was used to induce antigen-specific CD8＋ CTL. The apoptosis of L02 cells and HepG2 cells was detected after treatment with CD8＋ CTL. Interferon （IFN）-γ of CD8＋ CTL was detected by enzyme-linked immunospot assay （ELISPOT）. Subcutaneous tumor model of mouse HCC was established. The tumor volume of different groups was measured. Tumor tissue was further observed by pathological sections.Results MAGE-A3 protein stimulation significantly increased the levels of Human leukocyte antigen-DR （HLA-DR）, DC83, DC86 and DC80 markers on the surface of DC （97.45±2.58 vs. 48.33±1.68, 92.81±2.36 vs. 52.92±1.90, 94.00±3.01 vs. 53.97±2.05, 92.16±1.87 vs. 34.13±1.32; t=35.680, P=0.013; t=29.440, P=0.021; t=24.580, P=0.012; t=56.690, P=0.009）. The IL-12 level of MAGE-A3-DC was significantly higher than that of DC [（338.44±18.15） pg/ml vs. （243.23±16.56） pg/ml; t=8.670, P=0.005], but IL-10 level of MAGE-A3-DC was significantly lower than that of DC [（207.21±10.89） pg/ml vs. （327.58±14.36） pg/ml; t=14.930, P=0.009]. The apoptosis of L02 cells treated with MAGE-A3-DC-CD8＋ CTL was similar to that after treatment with DC-CD8＋ CTL [（9.10±1.40）% vs. （9.71±1.58）%; t=0.650, P=0.120]. The apoptostic rate of HepG2 cells treated with MAGE-A3-DC-CD8＋ CTL was significantly higher than that after treatment with DC-CD8＋ CTL [（58.84±5.27）% vs. （9.63±1.61）%; t=19.970, P=0.008]. After therapy, tumor sizes V/V0 in MAGE-A3-DC-CD8＋ CTL group decreased significantly as compared with those of phosphate buffer （PBS） group and DC-CD8＋ CTL group （5.43±1.22 vs. 21.81±2.01 vs. 22.85±2.40; t=22.03

关 键 词：癌 肝细胞 免疫治疗 黑色素瘤相关抗原-A3 树突状细胞 细胞毒T淋巴 细胞 

分 类 号：R735.7[医药卫生—肿瘤]