肾癌细胞和G250单克隆抗体复合物致敏树突状细胞肿瘤疫苗的制备

Preparation of dendritic cell-based cancer vaccines pulsed with G250 monoclonal antibody IgG-complexed renal carcinoma cells

目的:以肾癌细胞和G250单克隆抗体(G250 monoclonal antibody,G250 mAb)复合物致敏树突状细胞(dendriticcells,DC)制备肾癌DC疫苗,并检测其活化水平,为临床应用DC肿瘤疫苗提供依据。方法:制备凋亡的肾癌细胞与G250mAb形成的复合物(G250 mAb IgG-complexed apoptotic tumor cell,IC-ATC)。取健康人新鲜外周血分离单个核细胞(peripheralblood mononuclear cell,PBMC),以GM-CSF和IL-4诱导PBMC成未成熟的树突状细胞(immature dendritic cells,iDC),将iDC分别负载凋亡肿瘤细胞(apoptotic tumor cell,ATC)、IC-ATC、G250 mAb,并均以TNF-α诱导成熟树突状细胞(mature dendriticcells,mDC),将未负载的DC作为对照组。流式细胞术检测各组mDC的免疫表型、ELISA试剂盒检测IL-12分泌水平,CCK-8法检测mDC刺激淋巴细胞增殖的能力。结果:成功制备IC-ATC致敏的DC疫苗。相对于负载ATC、G250mAb和未负载的DC,负载IC-ATC的DC疫苗显著上调CD86、CD80、CD83、HLA-DR的表达[(42.04±3.42)%vs(28.34±1.16)%、(33.77±1.61)%、(26.52±2.14)%,P<0.05;(38.17±2.55)%vs(23.79±2.41)%、(31.94±3.29)%、(24.32±3.23)%,P<0.05;(79.39±1.44)%vs(69.06±2.01)%、(74.49±1.35)%、(66.71±3.83)%,P<0.05;(35.52±2.72)%vs(26.90±2.82)%、(29.45±1.58)%、(27.42±2.11)%,P<0.05]和IL-12分泌水平(25.04 vs 5.27、13.32、7.53,P<0.05),且能更有效地刺激淋巴细胞增殖(4.02 vs 1.73、1.22、1.41,P<0.01)。结论:IC-ATC可有效促进DC成熟和活化,IC-ATC致敏的DC疫苗诱导淋巴细胞增殖能力显著增强。

Objective： To use G250 monoclonal antibody（G250 mAb） IgG-complexed renal carcinoma cells to acti-vate dendritic cells（DCs）,develop the DC vaccine of renal carcinoma and determine its activation for clinical tumor biological therapy.Methods： Prepare apoptotic renal carcinoma cells and induce the G250 mAb-complexed apoptotic renal carcinoma cells（IC-ATC）.Immature dendritic cells（iDCs） induced from peripheral blood mononuclear cells of healthy volunteers were cultured and propagated in vitro using rhGM-CSF and rhIL-4.iDCs were loaded with apoptotic renal carcinoma cells（ATC）,IC-ATC and G250 mAb.Then mature dendritic cells（mDCs） were induced by TNF-α,while the DCs pulsed with no antigen served as a control group.The immune phenotype of mDCs in different groups was detected by flow cytometry,secretion of IL-12 by DCs was measured by ELISA and the ability of DCs to stimulate lymphocyte proliferation was examined by CCK-8 assay.Results： It was found that when compared with ATC,G250 mAb and the control group,the mDCs pulsed with IC-ATC obriously up-regulated the expressions of CD83,CD80,CD86,and HLA-DR（[42.04±3.42]% vs [28.34±1.16]%,[33.77±1.61]%,[26.52±2.14]%,P0.05;[38.17±2.55]% vs [23.79±2.41]%,[31.94±3.29]%,[24.32±3.23]%,P0.05;[79.39±1.44]% vs [69.06±2.01]%,[74.49±1.35]%,[66.71±3.83]%,P0.05;[35.52±2.72]% vs [26.90±2.82]%,[29.45±1.58]%,[27.42±2.11]%,P0.05）,and secreted higher quantity of IL-12（25.04 vs 5.27,13.32,7.53,P0.05）.Moreover,mDCs loaded by IC-ATC induced multiplication of lymphocytes was more effective（4.02 vs 1.73,1.22 vs 1.41,P0.05）.Conclusion： IC-ATC can promote DCs mature effectively,and DCs pulsed with IC-ATC can induce the proliferation and activation significantly.