转染A549细胞总RNA的树突状细胞疫苗抗肿瘤效应的体外研究  

Anti-tumor effect of dendritic cell vaccines transfected with total RNA of A549 cells in vitro

作　　者：完晓菊[1] 王保龙[1] 上官志敏[1] 叶书来[1] 陈家萍[1] 应美爱[1] 梁晓浏[1] 金娟[1] 王敏[1] 

机构地区：[1]安徽医科大学附属省立医院输血科,安徽合肥230001

出　　处：《中国输血杂志》2010年第1期19-24,共6页Chinese Journal of Blood Transfusion

基　　金：安徽省卫生厅医学科研课题(编号09A082)

摘　　要：目的探讨人肺腺癌细胞株A549总RNA转染的人树突状细胞(DCs)在体外诱导特异性抗肿瘤免疫反应的能力。方法利用密度梯度离心法从血细胞分离机新鲜采集的外周富集血中分离出单个核细胞(PBMCs),贴壁获得单核细胞,加入重组人粒细胞巨噬细胞集落刺激因子(rhGM-CSF)和重组人白细胞介素-4(rhIL-4)进一步诱导为树突状细胞(DCs),同时利用提取的A549细胞总RNA转染DCs来制备疫苗,并与T细胞混合培养诱导扩增细胞毒性T淋巴细胞(CTLs);实验分为转染A549细胞总RNA的DC组、未转染DC组和转染空脂质体DC组,分别以流式细胞术(FCM)鉴定DCs表型、酶联免疫吸附法(ELISA)检测IL-12、IFN-γ分泌水平、3H-TdR掺入检测T细胞增殖能力及乳酸脱氢酶(LDH)释放法测定杀伤肿瘤活性并比较各组差异。结果1)转染总RNA组DCs表面CD40、CD80、CD83、HLA-DR的表达较未转染组DCs和转染空脂质体组DCs均有升高,CD40在3组的表达率分别为(69.01±7.67)%、(19.28±3.51)%和(39.62±2.72)%;CD80在3组的表达率分别为:(74.39±6.46)%、(15.48±3.03)%和(25.70±2.92)%;CD83在3组的表达率分别为:(81.79±4.61)%、(13.29±2.34)%和(31.42±1.97)%;HLA-DR在3组的表达率分别为:(76.53±5.13)%、(49.23±4.05)%和(29.94±3.53)%,差异有统计学意义(P<0.05);2)转染总RNA组DCs的IL-12和IFN-γ分泌水平较未转染组及转染空脂质体组明显升高(P<0.05),3组IL-12分别为(543.24±68.33)、(77.11±27.65)和(167.78±31.84)pg/ml;3组IFN-γ分别为:(122.95±32.84)、(41.06±10.97)和(56.08±15.96)pg/ml;3)3H-TdR掺入试验所得的cpm,3组分别为(7437±720)、(3 807±476)和(4 543±719),阳性对照组为:16 739±91,差异有统计学意义(P<0.05);4)LDH释放法可见转染A549总RNA的DC组的特异性杀瘤活性(58.54±8.27)%,明显高于未转染组(36.03±4.03)%和转染空脂质体组(33.47±8.21)%,差异具有统计学意义(P<0.05)。结论人肺腺癌细胞A549总RNA转染的DC疫苗体外诱导的特异性抗肿瘤免疫能Objective To study the specific anti-tumor immune response of dendritic cells （DCs） transfected with human lung adenocarcinoma cell line A549 total RNA. Methods Using density gradient centrifugation method,the peripheral blood mononuclear cells （PBMCs） were isolated from fresh peripheral enriched blood, then obtain monocytes via making PBMCs adherent,DCs were generated from monocytes that induced by rhGM-CSF, rhIL-4, and transfected with total RNA extracted from A549 cells for preparing vaccine, and co-cultured with T cells to induce specific cytotoxic T lymphocyte （CTL）. DCs were divided into three groups to carry out the experiments. They were DCs transfected by liposome with tumor cell A549 total RNA, non-transfected DCs and liposome-transfected DCs. Surface markers on DC were detected by flow cytometry;the secretion of IL-12 and IFN-γ, was detected by enzyme-linked immunoassay（ELISA） ;T cell proliferation was judged by 3H-Tdr incorporation assay;the specific cytotoxicity was measured by lactate dehydrogenase （LDH） content. Results 1 ）Surface markers on total RNA-transfected DCs were expressed with remarkably higher levels than non-transfected and liposome-transfected DCs （ P 〈 0. 05 ）, CD40 expression rate in these three groups were （ 69. 01 ± 7.67 ） %, （ 19. 28 ± 3.51 ） % and （39.62 ± 2.72） % ; CD80 expression rate in these three groups were （74. 39 ± 6.46） %, （ 15.48 ± 3.03） % and （25.70 ± 2.92） % ; CD83 expression rate in these three groups were （ 81.79 ± 4.61 ） %, （ 13.29 ± 2. 34） %and （ 31.42 ±1.97） % ; HLA-DR expression rate in these three groups were （ 76.53 ± 5.13 ） %, （49. 23 ± 4.05 ） % and （29.94±3.53）% ;2）the secretion of IL-12 and IFN- γwas dramatically increased in RNA-transfected DCs thanother two groups （ P 〈 0.05 ）, IL-12 was （ 543.24 ± 68. 33 ） pg/ml, （ 77. 11 ± 27. 65 ） pg/ml and （ 167.78 ± 31.84 ） pg/ml ; IFN-γwas （ 122. 95± 32. 84） pg/ml, （41.06 ± 10. 97 ） pg/ml and �

关 键 词：人肺腺癌细胞(A549) 树突状细胞(DCs) RNA 转染 体外诱导 抗肿瘤疫苗 肿瘤免疫 

分 类 号：R446.6[医药卫生—诊断学]