【摘要】：目的：1.體外構建能夠有效激活T淋巴細胞抗腫瘤效應的DC疫苗體系；2.DC疫苗聯(lián)合VEGF單克隆抗體(Avastin Beva)協(xié)同治療口腔鱗癌的探索。方法：密度梯度離心法從外周血中分離單核細胞,細胞因子GM-CSF+IL-4+TNF-a聯(lián)合誘導,使單核細胞分化為成熟的功能性DC,觀察誘導過程中DC形態(tài)變化、檢測DC表面特異性表型分子、吞噬抗原能力、分泌IL-12水平及混合淋巴細胞增殖反應；反復凍融法制備口腔鱗癌細胞凍融抗原,與成熟DC共培養(yǎng)48h,致敏DC得到疫苗化DC；同時檢測DC疫苗特異性表型分子、吞噬抗原能力、分泌IL-12水平及混合淋巴細胞增殖反應。單核細胞貼壁過程中獲得懸浮淋巴細胞液,尼龍毛柱法分離T淋巴細胞,并與致敏的DC疫苗混合培養(yǎng)72h得到活化的細胞毒性T淋巴細胞(CTL)；檢測DC疫苗激活的CTL聯(lián)合VEGF單克隆抗體(Avastin Beva)的抗腫瘤效應。實驗分4組：單純口腔鱗癌細胞組；DC疫苗激活的T細胞組；VEGF單克隆抗體Avastin組：DC疫苗激活的T細胞+Avastin組；口腔鱗癌細胞CAL27細胞和SCC4細胞為靶細胞。CCK8孵育2h,檢測DC激活的CTL聯(lián)合Avastin對口腔鱗癌細胞的殺傷作用；ANNEXINV和PI雙染,流式細胞儀檢測口腔鱗癌細胞凋亡情況。結果：①單核細胞體外培養(yǎng)至第7天,可得到具有典型樹枝狀突起的DC, CD1a、CD83、HLA-DR、CD86、CD80、CD40表達率分別為29.46%、24.66%、28.58%、38.92%、2.60%、72.73%；培養(yǎng)第9天得到致敏DC疫苗,其CD1a、CD83、HLA-DR、CD86、CD80、CD40表達率分別為22.18%、28.73%、28.23%、50.68%、6.79%、79.55%。DC吞噬FITC-Dextran能力為45.20%,致敏后下降為26.71%；DC分泌IL-12水平為20.97pg/ml,致敏后分泌IL-12水平變?yōu)?7.58pg/ml；以極少量的DC即可刺激T淋巴細胞明顯增殖(1：100),致敏后DC疫苗刺激T淋巴細胞增殖能力增強。②DC疫苗激活的T細胞對口腔鱗癌CAL27細胞殺傷率為67.6%、其誘導的CAL27細胞的凋亡率為11.25%；聯(lián)合應用Avastin后殺傷率上升到74.80%和CAL27細胞凋亡率上升至17.02%,與單純應用DC疫苗組有顯著性差異(P0.05)。DC疫苗激活的T細胞對口腔鱗癌SCC4細胞殺傷率為66.7%、其誘導的SCC4細胞后的凋亡率為15.69%；聯(lián)合應用Avastin后殺傷率上升至75.10%和SCC4細胞凋亡率上升至23.85%,與單純應用DC疫苗組有顯著性差異(P0.05)。結論：成熟DC可以體外負載腫瘤抗原構建DC疫苗,該疫苗具有更強的抗原提呈能力；DC疫苗激活的T細胞聯(lián)合VEGF單克隆抗體(Avastin,Beva)對口腔鱗癌細胞有明顯的協(xié)同殺傷作用和促進口腔鱗癌細胞凋亡作用。
[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. 緇撴灉錛氣憼鍗曟牳緇嗚優(yōu)浣撳鍩瑰吇鑷崇7澶，

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