TLR4/MyD88信号通路在树突状细胞-肾癌786-0细胞株融合瘤苗中的作用  

Role of TLR4/MyD88 signal transduction pathway in fusion vaccine by peripheral blood dendritic cells and renal cell carcinoma 786-0 cells

作　　者：谭小军[1] 胡自力[1] 刘富[1] 

机构地区：[1]重庆医科大学附属第二医院泌尿外科,重庆400010

出　　处：《第三军医大学学报》2011年第15期1576-1581,共6页Journal of Third Military Medical University

摘　　要：目的探讨TOLL样受体4(toll-like receptor 4,TLR4)/髓样分化因子88(myeloid differentiation factor 88,MyD88)信号通路在树突状细胞(dentric cell,DC)-肾癌786-0细胞融合瘤苗中的变化及作用。方法用正常人外周血诱导不成熟的树突状细胞(immature dentric cell,imDC),利用聚乙二醇法(PEG)制成imDC-肾癌-786-0细胞融合瘤苗;用透射电镜观察imDC融合前后变化情况;RT-PCR检测单纯imDC组,肾癌786-0细胞组,imDC-肾癌-786-0融合瘤苗组在6、12、24、48 h中TLR4及MyD88 mRNA变化情况;用激光共聚焦检测imDC中核因子-κB(nuclear factor kappa B,NF-κB)在融合前后转位情况;流式细胞仪检测imDC在融合前后CD86、HLA-DR变化;噻唑盐(MTT)法检测各组体外刺激T淋巴细胞的增殖能力及刺激的细胞毒性T淋巴细胞(CTLs)的杀瘤活性。结果透射电镜显示融合细胞中imDC转变为mDC;TLR4及MyD88 mRNA在肾癌中不表达,在单纯imDC中呈低表达(TLR4/β-actin灰度比值0.40±0.03,MyD88/GAPDH灰度比值0.33±0.03),在融合瘤苗组中6 h表达最强(TLR4/β-actin灰度比值0.73±0.18,MyD88/GAPDH灰度比值为0.45±0.18),明显强于各组(P<0.05),此后在融合细胞中逐渐减弱,48 h基本不表达;激光共聚焦显示NF-κB在融合前位于imDC细胞质,融合后转移至细胞核;流式细胞仪提示融合瘤苗HLA-DR、CD86表达分别为81%、80%,明显高于imDC组(63%、59%)及肾癌细胞组(P<0.05);MTT法示融合疫苗在体外刺激T淋巴细胞增殖能力[D(570)值(1.95±0.22)]强于imDC组(1.31±0.35)及肾癌786-0组(1.28±0.33)(P<0.05),并且其诱导产生的CTLs对自体肾癌细胞具有显著的杀伤活性。结论 TLR4/MyD88-NF-κB信号通路在imDC与肾癌786-0细胞融合过程中促使树突状细胞成熟发挥了非常重要的作用,并且此信号通路能促使树突状细胞功能发生转变。Objective To investigate the change and function of toll-like receptor 4 （TLR4）/Myeloid differentiation factor 88 （MyD88） signal transduction pathway in fusion vaccine by peripheral blood dendritic cells and renal cell carcinoma 786-0 cells. Methods Peripheral blood sample of healthy volunteers was used to induce immature dendritic cells （imDCs） , which were further induced to fuse with renal cell carcinoma （RCC） 786-0 cells with aid of polyethylene glycol （PEG）. Transmission electron microscopy （TEM） was employed to observe the changes of imDCs before and after fusion. Semi-quantitative RT-PCR was used to detect the mRNA changes of TLR4 and MyD88 in pure imDCs, RCC 786-0 cells, and imDC-RCC 786-0 fusion vaccine in 6, 12, 24, and 48 h after fusion. The transposition of nuclear fac-torkappa B （ NF-κB ） in imDCs before and after fusion was detected by laser scanning eonfocal microscopy （LSCM）. The changes of CD86 and HLA-DR in imDCs before and after fusion were detected by flow cytometry. The abilities of the fusion vaccine to stimulate T lymphocytes proliferation and CTL cells-mediated antitumor response were measured by MTT assay. Results Transmission electron microscopy showed imDCs were transformed to mDCs after fusion. The mRNA of TLR4 and MyD88 was not expressed in RCC-786-0 cells, mildly expressed in imDCs, but was significantly strongly expressed in fusion vaccine in 6 h after fusion （P 〈 0.05 ）, then their mRNA expression was decreased gradually till undetectable in 48 h after fusion. LSCM displayed that NF-KB existed in the cytoplasm of pure imDC, and then translocated in the nucleus of fusion vaccine. The expressions of CD86 and HLA-DR in fusion vaccine were significantly upregulated compared with the pure imDC control group （ P 〈 0.05 ）. The functions of dendritic cell-tumor fusion vaccine to stimulate the proliferation of T lymphocytes were stronger than that of imDCs, RCC 786-0 cells （P 〈 0.05 ）, and CTLs stimulated by the fusion vaccine showed distin

关 键 词：TOLL样受体4 树突状细胞 肾癌 融合瘤苗 核转录因子 半定量RT—PCR 

分 类 号：R73-35[医药卫生—肿瘤] R730.23[医药卫生—临床医学]