论文部分内容阅读
目的:建立自体热休克凋亡食管癌细胞抗原制备、树突状细胞(DC)体外诱导以及抗原负载方法,为DC肿瘤疫苗的临床应用提供技术基础。方法:采用酶消化法从手术切除的食管癌新鲜组织获得单细胞悬液,热休克后用桦脂酸诱导其凋亡制备成细胞抗原;采用人工肝素抗凝采集外周静脉血,分离单个核细胞(PBMC),贴壁法获得单核细胞,经GM-CSF与IL-4体外诱导成未成熟树突状细胞(imDC);负载细胞抗原后制备成DC肿瘤疫苗,并对DC疫苗的形态、数量及表型特征进行分析。结果:肿瘤细胞抗原每克组织得率(9.89±2.46)×106,平均凋亡率(67.60±3.23)%。imDC活率>95%,imDC表型分析为CD11c+HLA-DR+、CD11c+CD80+、CD11c+CD83+和CD11c+CD86+,表达率分别为(88.30±3.59)%(、1.25±0.55)%、(6.13±1.79)%和(72.90±5.01)%。DC疫苗活率>95%,DC疫苗表型为CD11c+HLA-DR+、CD11c+CD80+、CD11c+CD83+和CD11c+CD86+,表达率分别为(89.60±3.91)%、(34.20±3.85)%、(64.90±8.05)%和(86.10±6.22)%。负载自体抗原DC诱导CTL杀伤自体肿瘤细胞的杀伤率为(65.90±6.25)%,明显高于杀伤食管癌肿瘤细胞株的杀伤率〔(32.56±2.68)%〕,P<0.01。结论:本方法稳定、安全及可靠,可制备出在体外诱导特异性CTL的DC肿瘤疫苗。
OBJECTIVE: To establish a method for the preparation of anti-apoptotic esophageal cancer cells, the induction of dendritic cells (DCs) and the method of antigen loading in esophageal cancer cells, providing the technical basis for the clinical application of DC tumor vaccines. Methods: The single cell suspension was obtained from freshly resected esophageal cancer tissue by enzymatic digestion method. After heat shock, the cells were induced to apoptosis by betulinic acid, and then the peripheral blood was obtained by anticoagulation with artificial heparin. Mononuclear cells (PBMC) and adherent method, immature DCs were induced by GM-CSF and IL-4 in vitro. DC vaccines were prepared after loading of cell antigens, and the morphology, Quantity and phenotypic characteristics of the analysis. Results: The tumor cell antigen yield per gram (9.89 ± 2.46) × 106, the average rate of apoptosis (67.60 ± 3.23)%. The imDC activity was> 95%. The imDC phenotype was (88.30 ± 3.59)% (1.25 ± 0.55)%, (6.13 ± 1.79)% and (72.90 ± 5.01)%. DC vaccine was> 95%, and the DC vaccine phenotype was (89.60 ± 3.91)%, (34.20 ± 3.85)% and (11.8 ± 3.85)% respectively for CD11c + HLA-DR +, CD11c + CD80 +, CD11c + CD83 + and CD11c + CD86 + 64.90 ± 8.05)% and (86.10 ± 6.22)% respectively. The killing rate of autologous tumor cells loaded with autoantigen DC induced by CTL was (65.90 ± 6.25)%, which was significantly higher than that of tumor cells (32.56 ± 2.68%) (P <0.01). Conclusion: The method is stable, safe and reliable and can be used to prepare DC tumor vaccine which induces specific CTL in vitro.