改良细胞因子鸡尾酒诱导肺腺癌细胞总RNA转染树突状细胞疫苗抗肿瘤效应的体内研究

Anti-tumor effect of dendritic cell vaccines transfected with total RNA of lung adenocarcinoma cell and induced by modified cytokine cocktail in vivo

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 cytokine cocktails on transplanted tumor-bearing nude mice. Methods Peripheral blood mononuclear cells（ PBMC） were first isolated from 38 peripheral blood specimens of normal donors who underwent PBSC collection and then were induced into immature DC. DC were transfected with total RNA of human lung adenocarcinoma cell line A549 through electroporation,then stimulated with conventional cytokine cocktail（ IL-6,IL-1β,TNF-α,PGE2） and modified cytokine cocktail（ IL-1 β,IL-6,TNF-α,PGE2,poly Ⅰ∶ C,Cp G ODN）,respectively. Antigen-sensitized DC were co-cultured with autologous T cells to activate antigen specific cytotoxic T lymphocyte（ CTL）. The phenotypes of T cells were analyzed by flow cytometry,and the secretion of IFN-γ,TNF-α,IL-4,IL-5 and IL-10 from T cells were measured by enzyme-linked immunosorbent assays（ ELISA）. After establishment of the A549 implanted lung adenocarcinoma model,nude mice were randomly divided into empty control group,immature group,conventional group and modified group（ n = 5）. Peritumoral injection of CTL was performed on day 15,day 22 and day 29 after inoculation of tumor cells. The volumes and weights of tumors were measured.The expression of COX-2,VEGF,Bcl-2 and Bax in the tumor tissues were determined using immunohistochemistry and Western blotting. Results（ 1） The percentage of CD3 ＋ CD8 ＋ T cells in immature group,conventional group and modified group were（ 35. 00 ± 3. 24） % vs（ 57. 10 ± 2. 69） % vs（ 63. 98 ± 1. 96） %（ P〈 0. 05）,respectively.（ 2） The secretion of IFN-γ in the above groups were（ 160. 12 ± 42. 01） pg / m L vs（ 263. 17 ± 55. 30） pg / m L vs（ 1 034. 30 ± 253.07） pg / m L（ P 〈0. 05）; the secretion of TNF-α in the above groups were（ 72. 72 ± 9. 13） pg / m L vs（ 65. 20 ± 8. 03）pg / m L vs（ 295. 89 ± 123. 80） pg / m L（ P〈 0. 05）; the secretion of IL-10 in the above groups were（ 7. 26 ± 1. 76） pg/ m L vs（ 31. 06 ± 4. 19） pg / m L vs（ 44. 01 ± 12. 12） pg / m L（ P 〈0. 05）. IL-4 and IL-5 in each group were undetectable,showing that T cells in modified group produced much higher levels of Th1 cytokines IFN-γ and TNF-α than immature group and conventional group.（ 3） After immunotherapy with CTL,tumor size of nude mice in immature group,conventional group and modified group were（ 512 ± 33） mm3vs（ 345 ± 63） mm^3vs（ 203 ± 52） mm^3,（ P 〈0. 05）,respectively.Moreover,the inhibitive ratio of tumor weight in modified group was the highest at 69. 62%.（ 4） The expression of Bax in modified group was significantly up-regulated and the expressions of COX-2,VEGF and Bcl-2 were down-regulated. Conclusion DC vaccines which are electroporated with total RNA of lung adenocarcinoma cell and induced by modified cytokine cocktail can effectively inhibit the growth of lung adenocarcinoma transplanted tumors in nude mice through significant induction on type Th1 immune response and production of effective antigen specific CTL.