树突状细胞疫苗联合VEGF单克隆抗体对口腔鳞癌细胞的作用研究
发布时间：2018-08-23 07:56
【摘要】：目的：1.体外构建能够有效激活T淋巴细胞抗肿瘤效应的DC疫苗体系；2.DC疫苗联合VEGF单克隆抗体(Avastin Beva)协同治疗口腔鳞癌的探索。方法：密度梯度离心法从外周血中分离单核细胞,细胞因子GM-CSF+IL-4+TNF-a联合诱导,使单核细胞分化为成熟的功能性DC,观察诱导过程中DC形态变化、检测DC表面特异性表型分子、吞噬抗原能力、分泌IL-12水平及混合淋巴细胞增殖反应；反复冻融法制备口腔鳞癌细胞冻融抗原,与成熟DC共培养48h,致敏DC得到疫苗化DC；同时检测DC疫苗特异性表型分子、吞噬抗原能力、分泌IL-12水平及混合淋巴细胞增殖反应。单核细胞贴壁过程中获得悬浮淋巴细胞液,尼龙毛柱法分离T淋巴细胞,并与致敏的DC疫苗混合培养72h得到活化的细胞毒性T淋巴细胞(CTL)；检测DC疫苗激活的CTL联合VEGF单克隆抗体(Avastin Beva)的抗肿瘤效应。实验分4组：单纯口腔鳞癌细胞组；DC疫苗激活的T细胞组；VEGF单克隆抗体Avastin组：DC疫苗激活的T细胞+Avastin组；口腔鳞癌细胞CAL27细胞和SCC4细胞为靶细胞。CCK8孵育2h,检测DC激活的CTL联合Avastin对口腔鳞癌细胞的杀伤作用；ANNEXINV和PI双染,流式细胞仪检测口腔鳞癌细胞凋亡情况。结果：①单核细胞体外培养至第7天,可得到具有典型树枝状突起的DC, CD1a、CD83、HLA-DR、CD86、CD80、CD40表达率分别为29.46%、24.66%、28.58%、38.92%、2.60%、72.73%；培养第9天得到致敏DC疫苗,其CD1a、CD83、HLA-DR、CD86、CD80、CD40表达率分别为22.18%、28.73%、28.23%、50.68%、6.79%、79.55%。DC吞噬FITC-Dextran能力为45.20%,致敏后下降为26.71%；DC分泌IL-12水平为20.97pg/ml,致敏后分泌IL-12水平变为37.58pg/ml；以极少量的DC即可刺激T淋巴细胞明显增殖(1：100),致敏后DC疫苗刺激T淋巴细胞增殖能力增强。②DC疫苗激活的T细胞对口腔鳞癌CAL27细胞杀伤率为67.6%、其诱导的CAL27细胞的凋亡率为11.25%；联合应用Avastin后杀伤率上升到74.80%和CAL27细胞凋亡率上升至17.02%,与单纯应用DC疫苗组有显著性差异(P0.05)。DC疫苗激活的T细胞对口腔鳞癌SCC4细胞杀伤率为66.7%、其诱导的SCC4细胞后的凋亡率为15.69%；联合应用Avastin后杀伤率上升至75.10%和SCC4细胞凋亡率上升至23.85%,与单纯应用DC疫苗组有显著性差异(P0.05)。结论：成熟DC可以体外负载肿瘤抗原构建DC疫苗,该疫苗具有更强的抗原提呈能力；DC疫苗激活的T细胞联合VEGF单克隆抗体(Avastin,Beva)对口腔鳞癌细胞有明显的协同杀伤作用和促进口腔鳞癌细胞凋亡作用。
[Abstract]:Purpose 1. Construction of DC vaccine system which can effectively activate T lymphocyte anti-tumor effect in vitro. 2. Co treatment of oral squamous cell carcinoma (OSCC) with DC vaccine combined with VEGF monoclonal antibody (Avastin Beva). Methods: monocytes were isolated from peripheral blood by density gradient centrifugation. Monocytes were induced by cytokine GM-CSF IL-4 TNF-a to differentiate into mature functional DCs. The morphological changes of DC were observed and the surface specific phenotypic molecules were detected. Phagocytic ability, secretion of IL-12 level and proliferation of mixed lymphocytes, preparation of frozen and thawed oral squamous cell antigen by repeated freezing and thawing method, co-culture with mature DC for 48 h to sensitize DC to vaccinated DC, detection of specific phenotypic molecules of DC vaccine, Phagocytosis, secretion of IL-12 and mixed lymphocyte proliferation. Suspension lymphocyte fluid was obtained during monocyte adhesion, T lymphocytes were separated by nylon hair column method and co-cultured with sensitized DC vaccine for 72 hours to obtain activated cytotoxic T lymphocyte (CTL);. To detect the anti-tumor effect of DC vaccine activated CTL combined with VEGF monoclonal antibody (Avastin Beva). The experiment was divided into four groups: the T cell group was activated by DC vaccine and the T cell Avastin group was activated by Avastin; Oral squamous cell CAL27 cells and SCC4 cells were incubated for 2 hours. The killing effect of DC activated CTL combined with Avastin on oral squamous carcinoma cells was detected by ANNEXINV and Pi staining. Apoptosis of oral squamous cell carcinoma cells was detected by flow cytometry. 缁撴灉锛氣憼鍗曟牳缁嗚優浣撳鍩瑰吇鑷崇7澶，
本文编号：2198387
[Abstract]:Purpose 1. Construction of DC vaccine system which can effectively activate T lymphocyte anti-tumor effect in vitro. 2. Co treatment of oral squamous cell carcinoma (OSCC) with DC vaccine combined with VEGF monoclonal antibody (Avastin Beva). Methods: monocytes were isolated from peripheral blood by density gradient centrifugation. Monocytes were induced by cytokine GM-CSF IL-4 TNF-a to differentiate into mature functional DCs. The morphological changes of DC were observed and the surface specific phenotypic molecules were detected. Phagocytic ability, secretion of IL-12 level and proliferation of mixed lymphocytes, preparation of frozen and thawed oral squamous cell antigen by repeated freezing and thawing method, co-culture with mature DC for 48 h to sensitize DC to vaccinated DC, detection of specific phenotypic molecules of DC vaccine, Phagocytosis, secretion of IL-12 and mixed lymphocyte proliferation. Suspension lymphocyte fluid was obtained during monocyte adhesion, T lymphocytes were separated by nylon hair column method and co-cultured with sensitized DC vaccine for 72 hours to obtain activated cytotoxic T lymphocyte (CTL);. To detect the anti-tumor effect of DC vaccine activated CTL combined with VEGF monoclonal antibody (Avastin Beva). The experiment was divided into four groups: the T cell group was activated by DC vaccine and the T cell Avastin group was activated by Avastin; Oral squamous cell CAL27 cells and SCC4 cells were incubated for 2 hours. The killing effect of DC activated CTL combined with Avastin on oral squamous carcinoma cells was detected by ANNEXINV and Pi staining. Apoptosis of oral squamous cell carcinoma cells was detected by flow cytometry. 缁撴灉锛氣憼鍗曟牳缁嗚優浣撳鍩瑰吇鑷崇7澶，
本文编号：2198387
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