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目的建立自体热休克凋亡大肠癌细胞抗原制备、树突状细胞(DC)体外诱导、以及抗原负载方法。方法采用酶消化法从手术切除的肠癌新鲜组织获得单细胞悬液,热休克后用桦酯酸诱导其凋亡,制备成细胞抗原;采集外周静脉血,分离单核细胞(PBMC),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(1)肿瘤细胞抗原得率:(11.87±0.66)×106/g组织;平均凋亡率:(93.13±2.3)%;(2)imDC平均得率为(9.73±0.84)×106/(121.64)×106个PBMC,活率>95%;imDC表型分析:CD11c+CD14-、CD11c+HLA-DR+、CD11c+CD80+、CD11c+CD83+、CD11c+CD86+表达率分别为(87.48±2.59)%、(87.92±0.97)%、(2.20±0.67)%、(4.86±1.21)%、(5.00±0.97)%;(3)DC平均得率为(6.74±0.97)×106/(9.73±0.84)×106个imDC,活率>95%,DC表型:CD11c+CD14-、CD11c+HLA-DR+、CD11c+CD80+、CD11c+CD83+、CD11c+CD86+表达率分别为(93.55±1.24)%、(89.77±1.35)%、(87.80±1.63)%、(70.64±6.42)%、(95.44±2.16)%。结论自体大肠癌细胞负载树突状细胞的方法稳定、安全、可靠,可制备出成熟DC。
OBJECTIVE: To establish the preparation of anti-apoptotic colorectal cancer cell antigen, induction of dendritic cells (DC) in vitro, and antigen loading method. Methods Single cell suspension was obtained from freshly resected colon cancer tissues by enzymatic digestion. After heat shock, the cells were induced to apoptosis by betulinic acid to prepare cell antigens. Peripheral venous blood was collected and mononuclear cells (PBMCs) were isolated. GM-CSF and IL-4 induced immature dendritic cells (imDC) in vitro; DC vaccine was prepared after loading cell antigen, and the morphological, quantitative and phenotypic characteristics of DC vaccine were analyzed. Results (1) The rate of tumor cell antigen was (11.87 ± 0.66) × 106 / g, the average apoptosis rate was (93.13 ± 2.3)%. (2) The average yield of imDC was (9.73 ± 0.84) × 106 / 121.64) × 106 PBMCs, the viability was> 95%. The imDC phenotype analysis showed that the expression rates of CD11c + CD14-, CD11c + HLA-DR +, CD11c + CD80 +, CD11c + CD83 + and CD11c + CD86 + were 87.48 ± 2.59% , (87.92 ± 0.97)%, (2.20 ± 0.67)%, (4.86 ± 1.21)%, (5.00 ± 0.97)%, respectively; (3) The average DC yield was 6.74 ± 0.97 × 106 / (9.73 ± 0.84) (93.55 ± 1.24)% and (89.77)%, respectively. The expression of CD11c + CD14-, CD11c + HLA-DR +, CD11c + CD80 +, CD11c + CD83 + and CD11c + ± 1.35%, (87.80 ± 1.63)%, (70.64 ± 6.42)% and (95.44 ± 2.16)%, respectively. Conclusion The method of autologous colorectal cancer cells loaded with dendritic cells is stable, safe and reliable, and can produce mature DCs.