【摘要】：目的：1.體外構(gòu)建能夠有效激活T淋巴細(xì)胞抗腫瘤效應(yīng)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(xì)胞,細(xì)胞因子GM-CSF+IL-4+TNF-a聯(lián)合誘導(dǎo),使單核細(xì)胞分化為成熟的功能性DC,觀察誘導(dǎo)過程中DC形態(tài)變化、檢測(cè)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(xì)胞增殖反應(yīng)；反復(fù)凍融法制備口腔鱗癌細(xì)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(shí)檢測(cè)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(xì)胞增殖反應(yīng)。單核細(xì)胞貼壁過程中獲得懸浮淋巴細(xì)胞液,尼龍毛柱法分離T淋巴細(xì)胞,并與致敏的DC疫苗混合培養(yǎng)72h得到活化的細(xì)胞毒性T淋巴細(xì)胞(CTL)；檢測(cè)DC疫苗激活的CTL聯(lián)合VEGF單克隆抗體(Avastin Beva)的抗腫瘤效應(yīng)。實(shí)驗(yàn)分4組：?jiǎn)渭兛谇击[癌細(xì)胞組；DC疫苗激活的T細(xì)胞組；VEGF單克隆抗體Avastin組：DC疫苗激活的T細(xì)胞+Avastin組；口腔鱗癌細(xì)胞CAL27細(xì)胞和SCC4細(xì)胞為靶細(xì)胞。CCK8孵育2h,檢測(cè)DC激活的CTL聯(lián)合Avastin對(duì)口腔鱗癌細(xì)胞的殺傷作用；ANNEXINV和PI雙染,流式細(xì)胞儀檢測(cè)口腔鱗癌細(xì)胞凋亡情況。結(jié)果：①單核細(xì)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(dá)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(dá)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(xì)胞明顯增殖(1：100),致敏后DC疫苗刺激T淋巴細(xì)胞增殖能力增強(qiáng)。②DC疫苗激活的T細(xì)胞對(duì)口腔鱗癌CAL27細(xì)胞殺傷率為67.6%、其誘導(dǎo)的CAL27細(xì)胞的凋亡率為11.25%；聯(lián)合應(yīng)用Avastin后殺傷率上升到74.80%和CAL27細(xì)胞凋亡率上升至17.02%,與單純應(yīng)用DC疫苗組有顯著性差異(P0.05)。DC疫苗激活的T細(xì)胞對(duì)口腔鱗癌SCC4細(xì)胞殺傷率為66.7%、其誘導(dǎo)的SCC4細(xì)胞后的凋亡率為15.69%；聯(lián)合應(yīng)用Avastin后殺傷率上升至75.10%和SCC4細(xì)胞凋亡率上升至23.85%,與單純應(yīng)用DC疫苗組有顯著性差異(P0.05)。結(jié)論：成熟DC可以體外負(fù)載腫瘤抗原構(gòu)建DC疫苗,該疫苗具有更強(qiáng)的抗原提呈能力；DC疫苗激活的T細(xì)胞聯(lián)合VEGF單克隆抗體(Avastin,Beva)對(duì)口腔鱗癌細(xì)胞有明顯的協(xié)同殺傷作用和促進(jìn)口腔鱗癌細(xì)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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