论文部分内容阅读
目的探讨不同细胞因子鸡尾酒诱导的树突状细胞(DC)疫苗对裸鼠移植瘤的抗肿瘤作用。方法从38名外周血干细胞移植术供者的外周血中分离单个核细胞(PBMC),诱导成未成熟DC,将人肺腺癌细胞A549总RNA电转染入DC,分别用传统细胞因子鸡尾酒(IL-6、IL-1β、TNF-α、PGE2)和改良细胞因子鸡尾酒(IL-1β、IL-6、TNF-α、PGE2、polyⅠ∶C、CpG ODN)刺激DC成熟。致敏DC与自体T细胞混合培养,诱导产生抗原特异性细胞毒T淋巴细胞(CTL),用流式细胞术鉴定T细胞表型,ELISA法检测IFN-γ、TNF-α、IL-4、IL-5和IL-10的分泌。建立肺腺癌A549荷瘤裸鼠模型,随机分为空白对照组、未成熟组、传统组和改良组(n=5),于肿瘤接种d15、d22、d29给予相应CTL治疗,测量肿瘤体积和重量,免疫组化法和Western blotting检测肿瘤组织COX-2、VEGF、Bcl-2和Bax蛋白表达水平。结果1)CD3+CD8+T细胞表达率(%):未成熟组、传统组和改良组分别为35.00±3.24 vs 57.10±2.69 vs 63.98±1.96(P<0.05);2)IFN-γ浓度(pg/mL):3组分别为160.12±42.01 vs 263.17±55.30 vs 1 034.30±253.07(P<0.05);TNF-α浓度(pg/mL):3组分别为72.72±9.13 vs 65.20±8.03 vs 295.89±123.80(P<0.05);IL-10浓度(pg/mL):3组分别为7.26±1.76 vs 31.06±4.19 vs 44.01±12.12(P<0.05);3组IL-4、IL-5含量均未检测出,可见改良组T细胞分泌Th1型细胞因子IFN-γ和TNF-α的能力较未成熟组和传统组明显增强;3)经CTL免疫治疗后,裸鼠肿瘤体积(mm3):3组分别为512±33 vs 345±63 vs203±52(P<0.05),且改良组抑瘤率最大,为69.62%;4)改良组DC诱导的CTL明显上调Bax的表达,下调COX-2、VEGF和Bcl-2的表达。结论改良细胞因子鸡尾酒诱导肺腺癌细胞总RNA转染的DC疫苗能诱导Th1型免疫应答及产生有效的抗原特异性CTL,对肺腺癌裸鼠移植瘤具有良好的抑制作用。
Objective To investigate the anti-tumor effect of dendritic cell (DC) vaccine induced by different cytokines on nude mice xenografts. Methods Mononuclear cells (PBMCs) were isolated from peripheral blood of 38 peripheral blood stem cell transplant recipients and induced into immature DCs. The total RNA of human lung adenocarcinoma cells A549 was electroporated into DCs and cultured with conventional cytokine cocktail (IL-6, IL-1β, TNF-α, PGE2) and modified cytokine cocktail (IL-1β, IL-6, TNF-α, PGE2, polyⅠ: C, CpG ODN) Induction of DCs and autologous T cells were mixed to induce the production of antigen-specific cytotoxic T lymphocytes (CTLs). The T cell phenotypes were identified by flow cytometry. The levels of IFN-γ, TNF-α, IL- IL-5 and IL-10 secretion. The lung adenocarcinoma A549 tumor-bearing nude mice model was established and randomly divided into blank control group, immature group, traditional group and modified group (n = 5). Corresponding CTL treatment was given on days of tumor inoculation on d15, d22 and d29. The expressions of COX-2, VEGF, Bcl-2 and Bax in tumor tissue were detected by immunohistochemistry and Western blotting. Results: 1) The expression rate of CD3 + CD8 + T cells in immature group was 35.00 ± 3.24 vs 57.10 ± 2.69 vs 63.98 ± 1.96 respectively (P <0.05); 2) The concentration of IFN- (pg / mL): 160.12 ± 42.01 vs 263.17 ± 55.30 vs 1 034.30 ± 253.07 (P <0.05); The levels of TNF-α in the three groups were 72.72 ± 9.13 vs 65.20 ± 8.03 vs 295.89 ± 123.80 (P <0.05). The levels of IL-10 and IL-10 in the three groups were 7.26 ± 1.76 vs 31.06 ± 4.19 vs 44.01 ± 12.12 (P <0.05) The results showed that T lymphocytes secreted Th1 cytokines IFN-γ and TNF-α in the modified group were significantly higher than those in the immature group and the traditional group. (3) The tumor volume (mm3) in nude mice after the CTL immunotherapy was 3 (P <0.05), and the inhibition rate was the highest in the modified group (69.62%); 4) The CTL induced by DC in the modified group significantly upregulated the expression of Bax and down-regulated the expression of COX-2, VEGF and Bcl-2 expression. Conclusion DC vaccine transfected with modified cytokine cocktail of total RNA of lung adenocarcinoma cells can induce Th1-type immune response and produce effective antigen-specific CTL, which has a good inhibitory effect on lung adenocarcinoma xenografts in nude mice.