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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 role of toll-like receptor 4 (TLR4) / myeloid differentiation factor 88 (MyD88) signaling pathway in dendritic cells (DCs) Fusion tumor vaccine changes and effects. Methods Immature dendritic cells (imDC) were induced by normal human peripheral blood and imDC-renal carcinoma-786-0 cells were fused by polyethylene glycol (PEG). Transmission electron microscopy The changes of TLR4 and MyD88 mRNA in imDC group, renal cancer 786-0 cell group and imDC-renal carcinoma-786-0 fusion tumor group at 6, 12, 24 and 48 h were detected by RT-PCR The translocation of nuclear factor kappa B (NF-κB) in imDC before and after fusion was detected by confocal laser scanning microscope. The changes of CD86 and HLA-DR in imDC before and after fusion were detected by flow cytometry. The proliferative ability of T lymphocytes stimulated in vitro and cytotoxic T lymphocytes (CTLs) in vitro were detected. RESULTS: Transmission electron microscopy revealed that the imDC was transformed into mDCs in fusion cells. TLR4 and MyD88 mRNA were not expressed in renal carcinoma but low in imDC (TLR4 / β-actin, 0.40 ± 0.03 and MyD88 / GAPDH, 0.33 ± 0.03). The expression level of TLR4 / β-actin was the highest at 6 h in fusion tumor vaccine group (0.73 ± 0.18, 0.45 ± 0.18 in MyD88 / GAPDH) , Then gradually weakened in the fusion cells and basically did not express after 48 h. Laser confocal analysis showed that NF-κB was located in the imDC cytoplasm before fusion and then transferred to the nucleus after fusion. Flow cytometry indicated that the expression of HLA-DR and CD86 (81%, 80%) were significantly higher than that of imDC group (63%, 59%) and renal cell carcinoma group (P <0.05). MTT assay showed that the fusion vaccine stimulated the proliferation of T lymphocytes in vitro [ 1.95 ± 0.22) was stronger than that of imDC group (1.31 ± 0.35) and renal cell carcinoma 786-0 group (1.28 ± 0.33) (P <0.05), and CTLs induced by it had significant cytotoxicity on autologous renal cancer cells. Conclusion The TLR4 / MyD88-NF-κB signaling pathway plays a very important role in promoting the maturation of dendritic cells during the fusion of imDC and 786-0 cells, and this signaling pathway can promote the dendritic cell function.